Role of Molecular Interactions and Oligomerization in Chaperone Activity of Recombinant Acr from Mycobacterium tuberculosis
Identifieur interne : 000D73 ( Pmc/Corpus ); précédent : 000D72; suivant : 000D74Role of Molecular Interactions and Oligomerization in Chaperone Activity of Recombinant Acr from Mycobacterium tuberculosis
Auteurs : Gautam Krishnan ; Utpal RoySource :
- Iranian Journal of Biotechnology [ 1728-3043 ] ; 2019.
Abstract
The chaperone activity of
The aim of this study was to establish the correlation of structure and function of recombinant Acr proteins both before and after gel filtration chromatography. The aim was also to find the oligomeric conformation of these samples and use this information to explain differences in activit.
The level of expression was 40 to 50 mg /l. The protein was expressed in a soluble form at 37°C and subsequently purified by a 3 step gradient of imidazole using Ni-NTA resin. Gel filtration chromatography showed recombinant Acr to be a mixture of 9 to 15-mers, whereas Native-PAGE analysis showed a large proportion of 5 and 7 mers in the non gel-filtered sample, while non gel –filtered samples showed more proportions of higher size oligomers. The chaperone activity of non gel-filtered (A) samples was less than gel-filtered (B) samples at 37°C with 24 µM required of A for complete inhibition as compared to 6 µM of B. The chaperone activity of non gel–filtered samples at 60°C showed complete inhibition of activity at a concentration of 44 µM. Molecular interaction studies showed influence of size of oligomers on molecular coverage of insulin B chain. Pre-heat treatment improved the activity only after the gel filtration.
The larger proportion of monomers in the non gel-filtered sample could explain the difference in activity as compared to the gel-filtered samples in terms of molecular interaction with insulin. Increased oligomer size favorably affected secondary structure, a finding not reported so far, and warranting further investigation. A molecular level interaction of inhibition was predicted using Avogadro number of molecules and oligomer size. The difference in activity after pre–heat treatment seemed to indicate an important role for oligomerization.
Url:
DOI: 10.229252/ijb.2370
PubMed: 32195287
PubMed Central: 7080971
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PMC:7080971Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Role of Molecular Interactions and Oligomerization in Chaperone Activity of Recombinant Acr from <italic>Mycobacterium tuberculosis</italic></title><author><name sortKey="Krishnan, Gautam" sort="Krishnan, Gautam" uniqKey="Krishnan G" first="Gautam" last="Krishnan">Gautam Krishnan</name><affiliation><nlm:aff id="aff1"> Department of Biological Sciences, BITS Pilani KK Birla Goa Campus, Zuari Nagar, Goa 403726, Goa, India</nlm:aff></affiliation></author><author><name sortKey="Roy, Utpal" sort="Roy, Utpal" uniqKey="Roy U" first="Utpal" last="Roy">Utpal Roy</name><affiliation><nlm:aff id="aff1"> Department of Biological Sciences, BITS Pilani KK Birla Goa Campus, Zuari Nagar, Goa 403726, Goa, India</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">32195287</idno><idno type="pmc">7080971</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080971</idno><idno type="RBID">PMC:7080971</idno><idno type="doi">10.229252/ijb.2370</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000D73</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000D73</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Role of Molecular Interactions and Oligomerization in Chaperone Activity of Recombinant Acr from <italic>Mycobacterium tuberculosis</italic></title><author><name sortKey="Krishnan, Gautam" sort="Krishnan, Gautam" uniqKey="Krishnan G" first="Gautam" last="Krishnan">Gautam Krishnan</name><affiliation><nlm:aff id="aff1"> Department of Biological Sciences, BITS Pilani KK Birla Goa Campus, Zuari Nagar, Goa 403726, Goa, India</nlm:aff></affiliation></author><author><name sortKey="Roy, Utpal" sort="Roy, Utpal" uniqKey="Roy U" first="Utpal" last="Roy">Utpal Roy</name><affiliation><nlm:aff id="aff1"> Department of Biological Sciences, BITS Pilani KK Birla Goa Campus, Zuari Nagar, Goa 403726, Goa, India</nlm:aff></affiliation></author></analytic><series><title level="j">Iranian Journal of Biotechnology</title><idno type="ISSN">1728-3043</idno><idno type="eISSN">2322-2921</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec id="st1"><title>Background:</title><p> The chaperone activity of <italic>Mycobacterium tuberculosis</italic> Acr is an important function that helps to prevent misfolding
of protein substrates inside the host, especially in conditions of hypoxia.</p></sec><sec id="st2"><title>Objectives:</title><p> The aim of this study was to establish the correlation of structure and function of recombinant Acr proteins both before and after gel filtration
chromatography. The aim was also to find the oligomeric conformation of these samples and use this information to explain differences in activit.</p></sec><sec id="st3"><title>Material and Methods:</title><p><italic>M. tuberculosis acr</italic> gene was cloned with an N-terminal His-tag in pET28a and expressed with IPTG induction in BL2 (DE3) competent
<italic>Escherichia coli</italic>. The activity of a recombinant Acr without gel filtration was checked by preventing thermal aggregation of citrate
synthase at 45°C and the chaperone activity against insulin B chain aggregation at 60°C and 37°C. On further purification using gel filtration
chromatography, the protein was again tested for chaperone activity using insulin as substrate at 37°C with two types of samples without and with
gel filtration designated A and B respectively. The effects of pre–heat treatment at 60 °C on chaperone activity of both A and B samples were studied
by performing the chaperone assay at 37°C.</p></sec><sec id="st4"><title>Results:</title><p> The level of expression was 40 to 50 mg /l. The protein was expressed in a soluble form at 37°C and subsequently purified by a 3 step gradient of imidazole
using Ni-NTA resin. Gel filtration chromatography showed recombinant Acr to be a mixture of 9 to 15-mers, whereas Native-PAGE analysis showed a large proportion
of 5 and 7 mers in the non gel-filtered sample, while non gel –filtered samples showed more proportions of higher size oligomers. The chaperone activity
of non gel-filtered (A) samples was less than gel-filtered (B) samples at 37°C with 24 µM required of A for complete inhibition as compared to 6 µM of B. The
chaperone activity of non gel–filtered samples at 60°C showed complete inhibition of activity at a concentration of 44 µM. Molecular interaction studies showed
influence of size of oligomers on molecular coverage of insulin B chain. Pre-heat treatment improved the activity only after the gel filtration.</p></sec><sec id="st5"><title>Conclusions:</title><p> The larger proportion of monomers in the non gel-filtered sample could explain the difference in activity as compared to the gel-filtered samples
in terms of molecular interaction with insulin. Increased oligomer size favorably affected secondary structure, a finding not reported so far, and warranting
further investigation. A molecular level interaction of inhibition was predicted using Avogadro number of molecules and oligomer size. The difference
in activity after pre–heat treatment seemed to indicate an important role for oligomerization.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Smith, I" uniqKey="Smith I">I Smith</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kaufmann, Sh" uniqKey="Kaufmann S">SH Kaufmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De, Jong W" uniqKey="De J">Jong W De</name></author><author><name sortKey="Leunissen, J" uniqKey="Leunissen J">J Leunissen</name></author><author><name sortKey="Voorter, Cem" uniqKey="Voorter C">CEM Voorter</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chang, Z" uniqKey="Chang Z">Z Chang</name></author><author><name sortKey="Primm, Tp" uniqKey="Primm T">TP Primm</name></author><author><name sortKey="Jakana, J" uniqKey="Jakana J">J Jakana</name></author><author><name sortKey="Lee, Ih" uniqKey="Lee I">IH Lee</name></author><author><name sortKey="Serysheva, I" uniqKey="Serysheva I">I Serysheva</name></author><author><name sortKey="Chiu, W" uniqKey="Chiu W">W Chiu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Valdez, Mm" uniqKey="Valdez M">MM Valdez</name></author><author><name sortKey="Clark, Ji" uniqKey="Clark J">JI Clark</name></author><author><name sortKey="Wu, Gj" uniqKey="Wu G">GJ Wu</name></author><author><name sortKey="Muchowski, Pj" uniqKey="Muchowski P">PJ Muchowski</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Preneta, R" uniqKey="Preneta R">R Preneta</name></author><author><name sortKey="Papavinasasundaram, Kg" uniqKey="Papavinasasundaram K">KG Papavinasasundaram</name></author><author><name sortKey="Cozzone, Aj" uniqKey="Cozzone A">AJ Cozzone</name></author><author><name sortKey="Duclos, B" uniqKey="Duclos B">B Duclos</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kennaway, Ck" uniqKey="Kennaway C">CK Kennaway</name></author><author><name sortKey="Benesch, Jl" uniqKey="Benesch J">JL Benesch</name></author><author><name sortKey="Gohlke, U" uniqKey="Gohlke U">U Gohlke</name></author><author><name sortKey="Wang, L" uniqKey="Wang L">L Wang</name></author><author><name sortKey="Robinson, Cv" uniqKey="Robinson C">CV Robinson</name></author><author><name sortKey="Orlova, Ev" uniqKey="Orlova E">EV Orlova</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Panda, Ak" uniqKey="Panda A">AK Panda</name></author><author><name sortKey="Chakraborty, A" uniqKey="Chakraborty A">A Chakraborty</name></author><author><name sortKey="Nandi, Sk" uniqKey="Nandi S">SK Nandi</name></author><author><name sortKey="Kaushik, A" uniqKey="Kaushik A">A Kaushik</name></author><author><name sortKey="Biswas, A" uniqKey="Biswas A">A Biswas</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yang, H" uniqKey="Yang H">H Yang</name></author><author><name sortKey="Huang, S" uniqKey="Huang S">S Huang</name></author><author><name sortKey="Dai, H" uniqKey="Dai H">H Dai</name></author><author><name sortKey="Gong, Y" uniqKey="Gong Y">Y Gong</name></author><author><name sortKey="Zheng, C" uniqKey="Zheng C">C Zheng</name></author><author><name sortKey="Chang, Z" uniqKey="Chang Z">Z Chang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gu, L" uniqKey="Gu L">L Gu</name></author><author><name sortKey="Abulimiti, A" uniqKey="Abulimiti A">A Abulimiti</name></author><author><name sortKey="Li, W" uniqKey="Li W">W Li</name></author><author><name sortKey="Chang, Z" uniqKey="Chang Z">Z Chang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Feng, X" uniqKey="Feng X">X Feng</name></author><author><name sortKey="Huang, S" uniqKey="Huang S">S Huang</name></author><author><name sortKey="Fu, X" uniqKey="Fu X">X Fu</name></author><author><name sortKey="Abulimiti, A" uniqKey="Abulimiti A">A Abulimiti</name></author><author><name sortKey="Chang, Z" uniqKey="Chang Z">Z Chang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fu, X" uniqKey="Fu X">X Fu</name></author><author><name sortKey="Jiao, W" uniqKey="Jiao W">W Jiao</name></author><author><name sortKey="Abulimiti, A" uniqKey="Abulimiti A">A Abulimiti</name></author><author><name sortKey="Chang, Z" uniqKey="Chang Z">Z Chang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mao, Q" uniqKey="Mao Q">Q Mao</name></author><author><name sortKey="Ke, D" uniqKey="Ke D">D Ke</name></author><author><name sortKey="Feng, X" uniqKey="Feng X">X Feng</name></author><author><name sortKey="Chang, Z" uniqKey="Chang Z">Z Chang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kurnellas, Mp" uniqKey="Kurnellas M">MP Kurnellas</name></author><author><name sortKey="Brownell, Se" uniqKey="Brownell S">SE Brownell</name></author><author><name sortKey="Su, L" uniqKey="Su L">L Su</name></author><author><name sortKey="Malkovskiy, Av" uniqKey="Malkovskiy A">AV Malkovskiy</name></author><author><name sortKey="Rajadas, J" uniqKey="Rajadas J">J Rajadas</name></author><author><name sortKey="Dolganov, G" uniqKey="Dolganov G">G Dolganov</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Soong, Jx" uniqKey="Soong J">JX Soong</name></author><author><name sortKey="Lim, Ts" uniqKey="Lim T">TS Lim</name></author><author><name sortKey="Choong, Ys" uniqKey="Choong Y">YS Choong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jee, B" uniqKey="Jee B">B Jee</name></author><author><name sortKey="Singh, Y" uniqKey="Singh Y">Y Singh</name></author><author><name sortKey="Yadav, R" uniqKey="Yadav R">R Yadav</name></author><author><name sortKey="Lang, F" uniqKey="Lang F">F Lang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Razavi, Ss" uniqKey="Razavi S">SS Razavi</name></author><author><name sortKey="Peyvandi, H" uniqKey="Peyvandi H">H Peyvandi</name></author><author><name sortKey="Badrkhani, Jam Ar" uniqKey="Badrkhani J">Jam AR Badrkhani</name></author><author><name sortKey="Safari, F" uniqKey="Safari F">F Safari</name></author><author><name sortKey="Teymourian, H" uniqKey="Teymourian H">H Teymourian</name></author><author><name sortKey="Mohajerani, Sa" uniqKey="Mohajerani S">SA Mohajerani</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Iran J Biotechnol</journal-id><journal-id journal-id-type="iso-abbrev">Iran. J. Biotechnol</journal-id><journal-title-group><journal-title>Iranian Journal of Biotechnology</journal-title></journal-title-group><issn pub-type="ppub">1728-3043</issn><issn pub-type="epub">2322-2921</issn><publisher><publisher-name>National Institute of Genetic Engineering and Biotechnology</publisher-name><publisher-loc>Iran</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">32195287</article-id><article-id pub-id-type="pmc">7080971</article-id><article-id pub-id-type="publisher-id">IJB-17-3</article-id><article-id pub-id-type="doi">10.229252/ijb.2370</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Role of Molecular Interactions and Oligomerization in Chaperone Activity of Recombinant Acr from <italic>Mycobacterium tuberculosis</italic></article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Krishnan</surname><given-names>Gautam</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Roy</surname><given-names>Utpal</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="aff1"><label>1</label> Department of Biological Sciences, BITS Pilani KK Birla Goa Campus, Zuari Nagar, Goa 403726, Goa, India</aff><author-notes><corresp id="cor1"><label>*</label><bold>Corresponding author:</bold>Utpal Roy, Department of Biological Sciences, BITS Pilani KK Birla Goa Campus, Zuari Nagar, Goa 403726, Goa, India. Tel: +90-9422390738, E-mail: <email>utpalroy010@gmail.com</email></corresp></author-notes><pub-date pub-type="collection"><month>9</month><year>2019</year></pub-date><pub-date pub-type="epub"><day>01</day><month>9</month><year>2019</year></pub-date><volume>17</volume><issue>3</issue><fpage>e2370</fpage><lpage>e2370</lpage><permissions><copyright-statement>Copyright: © 2019 The Author(s); Published by National Institute of Genetic Engineering and Biotechnology.</copyright-statement><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc/4.0/"><license-p>This is an open access article, distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License
(<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/4.0/">http://creativecommons.org/licenses/by-nc/4.0/</ext-link>)
which permits others to copy and redistribute material just in noncommercial usages, provided the original work is properly cited.</license-p></license></permissions><abstract><sec id="st1"><title>Background:</title><p> The chaperone activity of <italic>Mycobacterium tuberculosis</italic> Acr is an important function that helps to prevent misfolding
of protein substrates inside the host, especially in conditions of hypoxia.</p></sec><sec id="st2"><title>Objectives:</title><p> The aim of this study was to establish the correlation of structure and function of recombinant Acr proteins both before and after gel filtration
chromatography. The aim was also to find the oligomeric conformation of these samples and use this information to explain differences in activit.</p></sec><sec id="st3"><title>Material and Methods:</title><p><italic>M. tuberculosis acr</italic> gene was cloned with an N-terminal His-tag in pET28a and expressed with IPTG induction in BL2 (DE3) competent
<italic>Escherichia coli</italic>. The activity of a recombinant Acr without gel filtration was checked by preventing thermal aggregation of citrate
synthase at 45°C and the chaperone activity against insulin B chain aggregation at 60°C and 37°C. On further purification using gel filtration
chromatography, the protein was again tested for chaperone activity using insulin as substrate at 37°C with two types of samples without and with
gel filtration designated A and B respectively. The effects of pre–heat treatment at 60 °C on chaperone activity of both A and B samples were studied
by performing the chaperone assay at 37°C.</p></sec><sec id="st4"><title>Results:</title><p> The level of expression was 40 to 50 mg /l. The protein was expressed in a soluble form at 37°C and subsequently purified by a 3 step gradient of imidazole
using Ni-NTA resin. Gel filtration chromatography showed recombinant Acr to be a mixture of 9 to 15-mers, whereas Native-PAGE analysis showed a large proportion
of 5 and 7 mers in the non gel-filtered sample, while non gel –filtered samples showed more proportions of higher size oligomers. The chaperone activity
of non gel-filtered (A) samples was less than gel-filtered (B) samples at 37°C with 24 µM required of A for complete inhibition as compared to 6 µM of B. The
chaperone activity of non gel–filtered samples at 60°C showed complete inhibition of activity at a concentration of 44 µM. Molecular interaction studies showed
influence of size of oligomers on molecular coverage of insulin B chain. Pre-heat treatment improved the activity only after the gel filtration.</p></sec><sec id="st5"><title>Conclusions:</title><p> The larger proportion of monomers in the non gel-filtered sample could explain the difference in activity as compared to the gel-filtered samples
in terms of molecular interaction with insulin. Increased oligomer size favorably affected secondary structure, a finding not reported so far, and warranting
further investigation. A molecular level interaction of inhibition was predicted using Avogadro number of molecules and oligomer size. The difference
in activity after pre–heat treatment seemed to indicate an important role for oligomerization.</p></sec></abstract><kwd-group><kwd> Acr</kwd><kwd> Insulin</kwd><kwd> Chaperone</kwd><kwd> Oligomer</kwd><kwd> pre-heat Treatment</kwd><kwd><italic>Mycobacterium tuberculosis</italic></kwd></kwd-group></article-meta></front><body><sec sec-type="Background" id="sec1-1"><title>1. Background</title><p>Tuberculosis (TB) is the oldest disease known to mankind with the earliest cases reported through skeletal remains found 4,000 years ago. It is caused by <italic>Mycobacterium tuberculosis</italic> ( <xref rid="ref1" ref-type="bibr">1</xref>). Unlike other known microbes that are cleared from the body by the host immune response, <italic>M. tuberculosis</italic> persists in a caseous granuloma for many years, leading to the phenomenon of latent TB ( <xref rid="ref2" ref-type="bibr">2</xref>). The protein dominant in the latent phase, is alpha-crystallin (Acr), the hspX or hsp16.3 protein. Acr belongs to the family of small heat shock proteins in the size range of 12-43 kDa which are known to function as oligomers that prevent misfolding of other proteins ( <xref rid="ref3" ref-type="bibr">3</xref>).</p><p>Earlier reports on cloning and expression of <italic>M. tb</italic>. Acr, showed that, Acr molecules can be used for preventing aggregation of the substrates ( <xref rid="ref4" ref-type="bibr">4</xref>- <xref rid="ref8" ref-type="bibr">8</xref>). All Acr preparations entailed the use of a gel filtration step to maximize the activity of the protein. However, there is no documentation of the oligomeric status of the recombinant protein being used in the assays. It is assumed that Acr is functionally active as a trimer of trimers; or a dodecamer; which was initially reported in 2005 and recently confirmed in 2016 ( <xref rid="ref4" ref-type="bibr">4</xref>, <xref rid="ref7" ref-type="bibr">7</xref>, <xref rid="ref8" ref-type="bibr">8</xref>). However, none of these studies mentions the actual oligomeric state of the protein during the assay, the ratio of different size of monomers and about the molecular level interactions except for few reports ( <xref rid="ref9" ref-type="bibr">9</xref>). There are reports on the mechanism by which the protein binds to the substrate <italic>in vitro</italic> ( <xref rid="ref4" ref-type="bibr">4</xref>, <xref rid="ref10" ref-type="bibr">10</xref>- <xref rid="ref12" ref-type="bibr">12</xref>). It has been agreed that there is a dynamic association and dissociation of oligomers which helps in chaperone activity. Some interesting features of <italic>M.tb</italic> Acr include its ability to improve activity with pre- heat treatment due to increased exposure of hydrophobic surfaces to the substrate molecules that help them bind their substrates effectively ( <xref rid="ref9" ref-type="bibr">9</xref>). It has also been reported that pre-heating at 60°C enhances the activity due to a change in the secondary structure of β sheets and random coils ( <xref rid="ref10" ref-type="bibr">10</xref>, <xref rid="ref13" ref-type="bibr">13</xref>). However, few studies have been carried out so far on the interaction at the molecular level in terms of molecules binding to the substrate. Most of the assays in previous studies have used substrates like catalase, citrate synthase, alcohol dehydrogenase and insulin ( <xref rid="ref4" ref-type="bibr">4</xref>, <xref rid="ref8" ref-type="bibr">8</xref>- <xref rid="ref10" ref-type="bibr">10</xref>). In addition, all the assays reported with insulin B chain were carried out at 25°C or 37°C except for a few studies performed at higher temperatures ( <xref rid="ref10" ref-type="bibr">10</xref>, <xref rid="ref14" ref-type="bibr">14</xref>).</p></sec><sec sec-type="Objectives" id="sec1-2"><title>2. Objectives</title><p>We aimed to study the chaperone activity of Acr using citrate synthase as substrate at 45°C and insulin B chain as substrate at 60°C. At 60°C, a crucial energy barrier is overcome, allowing more hydrophobic surfaces to be exposed improving the activity of Acr ( <xref rid="ref13" ref-type="bibr">13</xref>). Here we report the findings on expression, purification, and activity of recombinant
<italic>M. tb</italic> Acr on the substrate insulin at both 37°C and 60°C. Very importantly we also investigated the difference in chaperone activity of Acr with insulin at 37°C with 2 different types of samples non-gel- filtered and gel filtered. Pre-heat treatment studies at 37°C and 60°C followed by insulin assay at 37°C was carried out to investigate the role of oligomers in activity.</p></sec><sec sec-type="Materials and Methods" id="sec1-3"><title>3. Materials and Methods</title><sec id="sec3-1"><title>3.1. Expression and Purification of Acr</title><p><italic>M. tb</italic> Acr was expressed as a full-length protein with N- terminal His-tag in pET28a. Two clones,
#3 and #6 were tested for expression in BL21DE3 cells by inducing at 37°C with 1mM Isopropyl β-D-1-thiogalactopyranoside (IPTG).
The protein was checked for soluble expression by lysing a small volume of induced cell pellet and analysing the supernatant
and pellet on Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). 1mM (IPTG) induced cell pellet
was lysed in 20mM Tris pH 7.0, 500 mM NaCl, 5% glycerol. The lysate was centrifuged at 20,000 g for 25 mins and the
supernatant loaded on to Ni-NTA column (Thermo Scientific, USA), washed with 10 and 100 mM imidazole in the same
buffer before eluting with a 3-step gradient of 300 mM, 400 mM and 500 mM imidazole in 20 mM Tris pH 7.0, 300 mM NaCl,
5% glycerol. The His-tag elutes were dialyzed against 20 mM Tris pH 7.0, 100 mM NaCl, 5% glycerol and checked for
chaperone activity against insulin B chain. These samples were designated as ‘<bold>A</bold>’ samples.</p></sec><sec id="sec3-2"><title>3.2. Gel Filtration of Acr</title><p>1.5 ml of the dialyzed His- tag elutes , equivalent to 3mg as was measured by the Bradford’s dye binding method was
loaded twice on Sephacryl S-200 HiPrep XK 16/70 column at a flow rate of 0.5 ml/min and the buffer passed for a total
volume of 1.5 column volumes using the Akta Purifier System. A 15% denaturing SDS-PAGE was run to analyze the peaks
obtained in the gel filtration runs. The gel-filtered elutes were designated as ‘<bold>B</bold>’ samples.</p></sec><sec id="sec3-3"><title>3.3. Native-PAGE Analysis</title><p>A Native-PAGE analysis was carried out for both non-gel-filtered and gel-filtered samples, designated as (A) and (B), respectively using 8-16% gradient Tris Glycine gel and Bovine Serum Albumin (BSA) as a standard marker. The oligomer size was estimated using a plot of Log molecular weight versus distance migrated and using the antilog to estimate the exact oligomeric size.</p></sec><sec id="sec3-4"><title>3.4. Insulin Aggregation Assay</title><p>The chaperone activities of non-gel-filtered and gel- filtered Acr samples were checked using the 1,4- dithiothreitol (DTT) induced aggregation assay using 1.0 mg/ml insulin in buffer (20 mM Tris pH 7.0, 100 mM NaCl and 5% glycerol) at 60°C at 360 nm over 20 mins. Insulin R injection (BIOCON, India) was concentrated from a stock of 0.14 mg/ml to 1.0 mg/ml using 3kDa concentrators from Amicon (Millipore). Insulin in buffer without DTT and insulin with 25 µM DTT were used as controls. All the components except DTT were added to all these samples, the absorbance values were auto zeroed, baseline adjustment was done from 350 nm to 370 nm and 25 mM DTT added to start aggregation. Acr was used at 4 different concentrations of 12, 22, 39 and 44 µM in 3 different reaction volumes of 250, 300 and 400 µl. Likewise, chaperone activity was carried out by preventing DTT induced aggregation of insulin B chain concentration at 118 μM at the wavelength of 360 nm over 30 mins at 37°C. Aggregation was started by adding 25 mM DTT and the absorbance recorded to look for inhibition of aggregation on addition of Acr. Two types of samples were used for the enzyme assays: (A) Non gel-filtered and (B) gel-filtered.</p><p>The activity of A samples was checked at concentrations of 6 and 24 μM and B samples at 1, 3 and 4 μM using the assay buffer 20 mM Tris pH 7.0, 100 mM NaCl, 5% glycerol.</p></sec><sec id="sec3-5"><title>3.5. Thermal Aggregation Assay</title><p>It was carried out using A sample alone at a concentration of 5 μM and 2.5 μM citrate synthase substrate (Sigma) in a reaction volume of 300 μl. The assay buffer used was 20 mM Tris pH 7.0, 100 mM NaCl, 5% glycerol. The protein was added to citrate synthase and the assay was carried out at 45 °C by measuring absorbance at 360 nm over 30 mins.</p></sec><sec id="sec3-6"><title>3.6. Heat Treatment Assays</title><p>The recombinant Acr, after purification by gel filtration, was assayed at 37°C at a lower concentration of 18 µM to observe inhibition of insulin (1mg/ml). After pre- heat treatment at 60°C for 15 mins, the assay was repeated to check for chaperone activity. Another batch of His-tag purified protein was assayed at 37°C at 11 µM to check inhibition of insulin aggregation (1 mg/ml). After pre-heat treatment of samples at 60°C for 15 mins, the assay was repeated to check for chaperone activity.</p></sec><sec id="sec3-7"><title>3.7. Molecular Level Calculations</title><p>The molecular level calculations were done using the<ext-link ext-link-type="uri" xlink:href="http://molbiol.edu.ru/eng/scripts/01_04.html">http://molbiol.edu.ru/eng/scripts/01_04.html</ext-link></p></sec></sec><sec sec-type="Results" id="sec1-4"><title>4. Results</title><sec id="sec4-1"><title>4.1. Expression and Purification of Acr</title><p>The recombinant clone #3 was used for further studies because of its higher level of expression, which varied from 40-50 mg/l as determined
by the Bradford’s dye binding assay (<xref ref-type="fig" rid="IJB-17-e2370-g001.tif">Fig. 1a</xref>). The purification using the 3- step elution with imidazole gradient showed greater
than 95% purity in the 500 mM imidazole fraction (<xref ref-type="fig" rid="IJB-17-e2370-g001.tif">Fig. 1b</xref>).
</p><fig id="IJB-17-e2370-g001.tif" position="float"><label>Figure 1</label><caption><p><bold>A</bold>) SDS-PAGE of Acr-pET28a expression: Lane 1, Marker 97, 66, 45, 30, 21, 14 kDa: Lane 2: uninduced vector; Lane 3:
induced vector; Lane 4: induced clone #3; Lane 5: uninduced clone #3; Lane 6: induced clone #6; Lane 7: uninduced
clone #6; Lane 8: Marker 97, 66, 45, 30, 21, 14 kDa. <bold>B</bold>) Lane 1: His-tag eluted fractions E1-E4; Lane 2: E5-E8;
Lane 3,4: E9-E12; Lane 5: Marker (3, 6, 14, 21, 30, 43 kDa); Lane 6: Wash; Lane 7: Flow through; Lane 8:
Sonicate supernatant; Lane 9: Sonicate pellet; Lane 10: Induced Acr. <bold>C</bold>) Gel Filtration Run 2 Chromatogram:
X Axis: UV 280 nm; Y Axis: Elution time (mins). <bold>D</bold>) Bio-Rad Standards Chromatogram: X Axis: UV 280 nm;
Y Axis: Elution time (mins) A - Aggregates + Thyroglobulin 670 kDa, 36.5 ml (73 mins); B - Globulin 158 kDa,
44 ml (88 mins); C - Ovalbumin 44 kDa, 55 ml (110 mins); D - Myoglobin 17 kDa, 77 ml (154 mins); E - vitamin B12 1.5 kDa,
115 ml (230 mins). <bold>E</bold>) Plot of Log Mol wt versus distance migrated. The distance travelled by the three BSA monomers
was plotted against the log molecular weight. <bold>F</bold>) 15% SDS-PAGE of gel filtration run: Lane 1: Gel filtered eluted
fractions B2; Lane 2: B1; Lane 3: C1; Lane 4: C2; Lane 5: C3; Lane 6: C4; Lane 8: Marker (10, 20, 30, 40, 50, 60, 70, 85, 100, 150, 200 kDa);
Lane 9, 10: Load of gel filtration run 10 and 30 μl; Lane 11,12: His-tag eluted sample (concentrated) 10 and 30 μl. <bold>G</bold>)
Native-PAGE analysis of His-tag elutes and gel-filtered elutes. Lane 1: Empty; Lane 2: BSA; Lane 4, 5: His-tag elutes 10
and 20 μl; Lane 6, 7, 8: Gel-filtered elutes 20, 20 and 40 μl; Lane 9: BSA; Lane 11, 12: Gel-filtered elutes 20 and 40 μl Lane 13, 14: His-tag elutes 7 and 15 μl.</p></caption><graphic xlink:href="IJB-17-e2370-g001"></graphic></fig></sec><sec id="sec4-2"><title>4.2. Gel Filtration of Acr</title><p>In the 1<sup>st</sup> run, a major peak was observed at 37.5 ml (75 mins) while the peak ended at 44 ml (88 mins),
the position corresponding to the globulin peak of 158 kDa (data not shown). In the 2<sup>nd</sup> run, a peak was observed at
the same time while it again ended at 44 ml. A few smaller peaks were observed later (<xref ref-type="fig" rid="IJB-17-e2370-g001.tif">Fig. 1c</xref>).
</p><p> The Sephacryl S-200 column was calibrated with BioRad standards (Catalogue no.151-1901), aggregates with thyroglobulin 670 kDa,
globulin 158 kDa, ovalbumin 44 kDa, myoglobin 17 kDa and vitamin B12 1.5 kDa (<xref ref-type="fig" rid="IJB-17-e2370-g001.tif">Fig. 1d</xref>). A 15% denaturing SDS-PAGE was run
to analyze the peak obtained throughthe gel filtration runs (<xref ref-type="fig" rid="IJB-17-e2370-g001.tif">Fig. 1e</xref>). Acr appeared as an oligomer from the preliminary
data, with a size between 158 kDa and 670 kDa. No protein was observed in the later peaks.
</p></sec><sec id="sec4-3"><title>4.3. Native-PAGE Analysis</title><p>The Native-PAGE analysis showed a predominance of 9 to 15 mers with a higher amount of 5 and 7 mers in the case of the A samples (<xref ref-type="fig" rid="IJB-17-e2370-g001.tif">Fig. 1f</xref>).
The oligomeric calculation was performed by comparing the distance migrated by the oligomers with the BSA
standards (<xref ref-type="fig" rid="IJB-17-e2370-g001.tif">Fig. 1g</xref>, <xref rid="T1" ref-type="table">Table 1</xref>)</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p> Oligomer sizes have been calculated using BSA monomers, dimers and trimers as a reference (66, 132 and 198 kDa) and the log of molecular weight plotted versus the distance travelled in centimeter. From this calculation, the molecular weight has been extrapolated for the different bands observed in A (band 1 to 8) and B samples (band 1 to 5).</p></caption><table frame="hsides" rules="groups" width="100%"><thead><tr><th align="left" colspan="1" rowspan="1" valign="top">BSA (Mol. wt.)</th><th align="left" colspan="1" rowspan="1" valign="top">Log Mol wt.</th><th align="left" colspan="1" rowspan="1" valign="top">Distance (cm)</th><th align="left" colspan="3" rowspan="1" valign="top"></th></tr></thead><tbody><tr><td align="left" colspan="1" rowspan="1" valign="top">66</td><td align="left" colspan="1" rowspan="1" valign="top">1.82</td><td align="left" colspan="3" rowspan="1" valign="top">2</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top">132</td><td align="left" colspan="1" rowspan="1" valign="top">2.12</td><td align="left" colspan="2" rowspan="1" valign="top">5</td><td align="left" colspan="2" rowspan="1" valign="top">Monomer</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top">198</td><td align="left" colspan="1" rowspan="1" valign="top">2.29</td><td align="left" colspan="2" rowspan="1" valign="top">7</td><td align="left" colspan="2" rowspan="1" valign="top">18</td></tr><tr><td align="left" colspan="3" rowspan="1" valign="top"></td><td align="left" colspan="1" rowspan="1" valign="top">Size (kDa)</td><td align="left" colspan="1" rowspan="1" valign="top">Oligomer size</td><td align="left" colspan="1" rowspan="1" valign="top">% Fraction of total</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>A band 1</bold></td><td align="left" colspan="1" rowspan="1" valign="top">3</td><td align="left" colspan="1" rowspan="1" valign="top">82</td><td align="left" colspan="1" rowspan="1" valign="top">5</td><td align="left" colspan="1" rowspan="1" valign="top">14</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>A band 2</bold></td><td align="left" colspan="1" rowspan="1" valign="top">5</td><td align="left" colspan="1" rowspan="1" valign="top">129.93</td><td align="left" colspan="1" rowspan="1" valign="top">7</td><td align="left" colspan="1" rowspan="1" valign="top">9</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>A band 3</bold></td><td align="left" colspan="1" rowspan="1" valign="top">6</td><td align="left" colspan="1" rowspan="1" valign="top">158.49</td><td align="left" colspan="1" rowspan="1" valign="top">9</td><td align="left" colspan="1" rowspan="1" valign="top">20</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>A band 4</bold></td><td align="left" colspan="1" rowspan="1" valign="top">6.5</td><td align="left" colspan="1" rowspan="1" valign="top">177.82</td><td align="left" colspan="1" rowspan="1" valign="top">10</td><td align="left" colspan="1" rowspan="1" valign="top">15</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>A band 5</bold></td><td align="left" colspan="1" rowspan="1" valign="top">7</td><td align="left" colspan="1" rowspan="1" valign="top">198.15</td><td align="left" colspan="1" rowspan="1" valign="top">11</td><td align="left" colspan="1" rowspan="1" valign="top">12</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>A band 6</bold></td><td align="left" colspan="1" rowspan="1" valign="top">7.5</td><td align="left" colspan="1" rowspan="1" valign="top">221.3</td><td align="left" colspan="1" rowspan="1" valign="top">12</td><td align="left" colspan="1" rowspan="1" valign="top">8</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>A band 7</bold></td><td align="left" colspan="1" rowspan="1" valign="top">8</td><td align="left" colspan="1" rowspan="1" valign="top">246.6</td><td align="left" colspan="1" rowspan="1" valign="top">14</td><td align="left" colspan="1" rowspan="1" valign="top">11</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>A band 8</bold></td><td align="left" colspan="1" rowspan="1" valign="top">8.5</td><td align="left" colspan="1" rowspan="1" valign="top">275.4</td><td align="left" colspan="2" rowspan="1" valign="top">15</td></tr><tr><td align="left" colspan="4" rowspan="1" valign="top"></td><td align="left" colspan="1" rowspan="1" valign="top">Ratio of 11 to 15 mer </td><td align="left" colspan="1" rowspan="1" valign="top"> 43</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>B band 1</bold></td><td align="left" colspan="1" rowspan="1" valign="top">6.5</td><td align="left" colspan="1" rowspan="1" valign="top">177.82</td><td align="left" colspan="1" rowspan="1" valign="top">9</td><td align="left" colspan="1" rowspan="1" valign="top">15</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>B band 2</bold></td><td align="left" colspan="1" rowspan="1" valign="top">7</td><td align="left" colspan="1" rowspan="1" valign="top">198.15</td><td align="left" colspan="1" rowspan="1" valign="top">10</td><td align="left" colspan="1" rowspan="1" valign="top">15</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>B band 3</bold></td><td align="left" colspan="1" rowspan="1" valign="top">7.5</td><td align="left" colspan="1" rowspan="1" valign="top">221.3</td><td align="left" colspan="1" rowspan="1" valign="top">11</td><td align="left" colspan="1" rowspan="1" valign="top">15</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>B band 4</bold></td><td align="left" colspan="1" rowspan="1" valign="top">8</td><td align="left" colspan="1" rowspan="1" valign="top">246.6</td><td align="left" colspan="1" rowspan="1" valign="top">12</td><td align="left" colspan="1" rowspan="1" valign="top">15</td></tr><tr><td align="left" colspan="2" rowspan="1" valign="top"><bold>B band 5</bold></td><td align="left" colspan="1" rowspan="1" valign="top">8.5</td><td align="left" colspan="1" rowspan="1" valign="top">275.4</td><td align="left" colspan="1" rowspan="1" valign="top">16</td><td align="left" colspan="1" rowspan="1" valign="top">40</td></tr><tr><td align="left" colspan="4" rowspan="1" valign="top"></td><td align="left" colspan="1" rowspan="1" valign="top">Ratio of 11 to 15 mer </td><td align="left" colspan="1" rowspan="1" valign="top"> 75</td></tr></tbody></table></table-wrap></sec><sec id="sec4-4"><title>4.4. Insulin Assays</title><p> Acr showed dose-dependent activity against insulin B chain at 60°C with 100% inhibition obtained at a mole ratio of 0.35 (<xref ref-type="fig" rid="IJB-17-e2370-g002.tif">Fig. 2a</xref>).
A clear trend of increase in inhibition was observed with increase in molar concentration of Acr (<xref rid="T2" ref-type="table">Table 2</xref>). The amount of Acr
molecules used to inhibit insulin was calculated in terms of number of molecules and it was found that this number ranged from
0.6 x 10<sup>16</sup> to 1.5 x 10<sup>16</sup> while the insulin B chain molecules ranged from 1.8 x 10<sup>15</sup> to 1.05 x 10<sup>16</sup>, wherein, more than 50% of
the insulin B chain was covered, and the complete inhibition of aggregation was achieved (<xref ref-type="fig" rid="IJB-17-e2370-g002.tif">Fig. 2a</xref>). At 37°C, the chaperone activity
of the A samples showed nearly 100% inhibition at a concentration of 24 µM while, the B samples showed 95% inhibition at a concentration of 6 µM (<xref ref-type="fig" rid="IJB-17-e2370-g002.tif">Fig. 2b, 2c</xref>).</p><fig id="IJB-17-e2370-g002.tif" position="float"><label>Figure 2</label><caption><p><bold>A</bold>) Chaperone assay of insulin aggregation (60°C) with non-gel-filtered samples:
The assay was carried out using 125 µM insulin in an assay volume of 400 µl and of non-gel-filtered (A)
and at Acr concentration of 44 µM along with controls of insulin without DTT and insulin with DTT.
<bold>B</bold>) Chaperone assay of insulin aggregation (37°C) with non-gel-filtered samples: The assay of prevention
of insulin B chain aggregation using A samples: insulin plus DTT; insulin with plus 6 μM of non-gel-filtered (A) sample; insulin with 24 μM of non-gel-filtered (A) sample.
<bold>C</bold>) Chaperone assay of insulin aggregation (37°C) with gel-filtered samples: The assay of prevention
of insulin B chain aggregation using B samples. Assay of prevention of insulin B chain aggregation using gel-filtered
(B) samples. Insulin with DTT; 2: insulin plus 1μM gel-filtered (B) samples; 3: insulin with 3μM gel-filtered (B) samples;
4: insulin with 4μM gel-filtered (B) samples. <bold>D</bold>) Assay of thermal aggregation of citrate synthase using non gel-filtered
(A) sample. Maroon dash indicates blank; light blue dash indicates buffer with citrate synthase light green dash indicates citrate synthase with Acr.
<bold>E</bold>) Chaperone assay of insulin aggregation (37°C) after pre-heat treatment of gel-filtered samples:
Inhibition of insulin aggregation using Acr- gel-filtered (B) sample (18 µM) and insulin 0.5 mg/ml was carried
out at 37ºC. Acr heated at 60ºC was used to inhibit aggregation of insulin using DTT. <bold>F</bold>) Chaperone assay of insulin
aggregation (37°C) after pre-heat treatment of non-gel-filtered samples: Inhibition of insulin aggregation using Acr-A sample (11µM)
without gel filtration and insulin 1 mg/ml at 37°C. Acr heated at 60°C was used to inhibit aggregation of insulin using DTT.
</p></caption><graphic xlink:href="IJB-17-e2370-g002"></graphic></fig><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Molecular level interaction of Acr with insulin B chain at 60°C: Acr at 4 different concentrations was assayed with insulin
at 60°C and the activity checked by measuring aggregation over 20 mins. The % inhibition measured was checked as
a function of final OD divided by the final OD (minus insulin).The number of Acr molecules have beencalculated
by entering the amount (μg) in the website <ext-link ext-link-type="uri" xlink:href="http://molbiol.edu.ru/eng/scripts/01_04.html">http://molbiol.edu.ru/eng/scripts/01_04.html.</ext-link></p></caption><table frame="hsides" rules="groups" width="100%"><thead><tr><th align="left" colspan="1" rowspan="1" valign="top">Mole ratio of Acr/insulin</th><th align="left" colspan="1" rowspan="1" valign="top">% inhibition</th><th align="left" colspan="1" rowspan="1" valign="top">% of insulin covered</th><th align="left" colspan="1" rowspan="1" valign="top">Conc. of Acr(mg/ml)</th><th align="left" colspan="1" rowspan="1" valign="top">Molecules of Acr </th><th align="left" colspan="1" rowspan="1" valign="top"> Molecules of 3 kDainsulin</th></tr></thead><tbody><tr><td align="left" colspan="1" rowspan="1" valign="top">0.144</td><td align="left" colspan="1" rowspan="1" valign="top">53</td><td align="left" colspan="1" rowspan="1" valign="top">14</td><td align="left" colspan="1" rowspan="1" valign="top">0.2</td><td align="left" colspan="1" rowspan="1" valign="top">1.8 x10<sup>15</sup></td><td align="left" colspan="1" rowspan="1" valign="top">1.26 x 10<sup>16</sup></td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top">0.26</td><td align="left" colspan="1" rowspan="1" valign="top">56</td><td align="left" colspan="1" rowspan="1" valign="top">47</td><td align="left" colspan="1" rowspan="1" valign="top">0.36</td><td align="left" colspan="1" rowspan="1" valign="top">4 x 10<sup>15</sup></td><td align="left" colspan="1" rowspan="1" valign="top">0.85 x 10<sup>16</sup></td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top">0.35</td><td align="left" colspan="1" rowspan="1" valign="top">100</td><td align="left" colspan="1" rowspan="1" valign="top">70</td><td align="left" colspan="1" rowspan="1" valign="top">0.8</td><td align="left" colspan="1" rowspan="1" valign="top">1.05 x 10<sup>16</sup></td><td align="left" colspan="1" rowspan="1" valign="top">1.5 x 10<sup>16</sup></td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top">0.46</td><td align="left" colspan="1" rowspan="1" valign="top">95</td><td align="left" colspan="1" rowspan="1" valign="top">94</td><td align="left" colspan="1" rowspan="1" valign="top">0.7</td><td align="left" colspan="1" rowspan="1" valign="top">5.9 x 10<sup>15</sup></td><td align="left" colspan="1" rowspan="1" valign="top">0.63 x 10<sup>16</sup></td></tr></tbody></table></table-wrap></sec><sec id="sec4-5"><title>4.5. Thermal Aggregation Assay</title><p>The Thermal aggregation assay showed complete inhibition of activity at mole ratios of 2:1 of Acr to citrate synthase (<xref ref-type="fig" rid="IJB-17-e2370-g002.tif">Fig. 2d</xref>).</p></sec><sec id="sec4-6"><title>4.6. Heat Treatment Assays</title><p>The gel-filtered sample showed complete inhibition of insulin B chain after pre–heat treatment at 60°C for 15 mins (<xref ref-type="fig" rid="IJB-17-e2370-g002.tif">Fig. 2e</xref>).
His-tag eluted sample failed to exhibit improvement in chaperone activity after pre-heat treatment, suggesting the need
for secondary structure analysis to explain this difference (<xref ref-type="fig" rid="IJB-17-e2370-g002.tif">Fig. 2f</xref>).</p></sec><sec id="sec4-7"><title>4.7. Molecular Level Calculations</title><p>The molecular level calculations showed a typical pattern. Even though the percentage of insulin B chain covered was less in the gel-filtered (B) samples,
the percentage inhibition was more than the non-gel- filtered (A) samples (<xref rid="T3" ref-type="table">Table 3</xref>, <xref rid="T4" ref-type="table">4</xref>,
<xref rid="T5" ref-type="table">5</xref>). The molecular level calculations
showed better coverage of insulin B chain if we assume Acr to be a nonamer instead of monomer (<xref ref-type="fig" rid="IJB-17-e2370-g003.tif">Fig. 3a, 3b</xref>and <xref ref-type="fig" rid="IJB-17-e2370-g003.tif">3c</xref>).
</p><fig id="IJB-17-e2370-g003.tif" position="float"><label>Figure 3</label><caption><p><bold>A</bold>) <xref rid="T2" ref-type="table">Table 2</xref> data has been used to replot the mole ratio of gel-filtered Acr to insulin B chain in terms
of percentage inhibition obtained. <bold>B</bold>) <xref rid="T2" ref-type="table">Table 2</xref> data has been used to replot the number of Acr molecules that binds
to insulin in terms of percentage of insulin B chain covered, assuming Acr to be an 18 kDa monomer and dividing
the Avogadro number of molecules of Acr by the number of molecules of insulin B chain versus percentage inhibition.
<bold>C</bold>) Chaperone assay of insulin aggregation (37°C) with gel-filtered samples: The assay of prevention of insulin B chain
aggregation using B samples. Assay of prevention of insulin B chain aggregation using gel-filtered (B) samples. Insulin with DTT; 2:
insulin plus 1μM gel-filtered (B) samples; 3: insulin with 3μM gel-filtered (B) samples; 4: insulin with 4μM gel-filtered (B) samples.
<bold>D</bold>) Assay of thermal aggregation of citrate synthase using non gel-filtered (A) sample. Maroon dash indicates blank;
light blue dash indicates buffer with citrate synthase light green dash indicates citrate synthase with Acr.
<bold>E</bold>) Chaperone assay of insulin aggregation (37°C) after pre-heat treatment of gel-filtered samples:
Inhibition of insulin aggregation using Acr- gel-filtered (B) sample (18 μM) and insulin 0.5 mg/ml was carried out at 37ºC.
Acr heated at 60ºC was used to inhibit aggregation of insulin using DTT.
<bold>F</bold>) Chaperone assay of insulin aggregation (37°C)
after pre-heat treatment of non-gel-filtered samples: Inhibition of insulin aggregation using Acr-A sample (11μM) without gel
filtration and insulin 1 mg/ml at 37°C. Acr heated at 60°C was used to inhibit aggregation of insulin using DTT.
</p></caption><graphic xlink:href="IJB-17-e2370-g003"></graphic></fig><table-wrap id="T3" position="float"><label>Table 3</label><caption><p> Molecular level interaction of <bold>A</bold> sample with insulin B chain. The assumption is that Acr is 18 kDa and the insulin B chain is 3 kDa.
The number of molecules of insulin has been calculated based on an assay volume of 0.25 ml and the number of insulin molecules assumed to be 1.08 x10<sup>16</sup>.</p></caption><table frame="hsides" rules="groups" width="100%"><thead><tr><th align="left" colspan="1" rowspan="1" valign="top">% insulin B chain covered</th><th align="left" colspan="1" rowspan="1" valign="top">% inhibition</th><th align="left" colspan="1" rowspan="1" valign="top">Molecules of Acr covered</th><th align="left" colspan="1" rowspan="1" valign="top">Acr concentration (μM)</th></tr></thead><tbody><tr><td align="left" colspan="1" rowspan="1" valign="top">1.68</td><td align="left" colspan="1" rowspan="1" valign="top">28</td><td align="left" colspan="1" rowspan="1" valign="top">1.81 x 10<sup>15</sup></td><td align="left" colspan="1" rowspan="1" valign="top">6</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top">3.37</td><td align="left" colspan="1" rowspan="1" valign="top">90</td><td align="left" colspan="1" rowspan="1" valign="top">3.64 x10<sup>15</sup></td><td align="left" colspan="1" rowspan="1" valign="top">24</td></tr></tbody></table></table-wrap><table-wrap id="T4" position="float"><label>Table 4</label><caption><p> Molecular level interaction of <bold>B</bold> sample with insulin B chain. The assumption is that Acr was 18 kDa and the insulin B chain was 3 kDa.
The number of molecules of insulin has been calculated based on an assay volume of 0.25 ml and the number of insulin molecules assumed to be 1.08 x 10<sup>16</sup>.</p></caption><table frame="hsides" rules="groups" width="100%"><thead><tr><th align="left" colspan="1" rowspan="1" valign="top">% insulin B chain covered</th><th align="left" colspan="1" rowspan="1" valign="top">% inhibition</th><th align="left" colspan="1" rowspan="1" valign="top">Molecules of Acr </th><th align="left" colspan="1" rowspan="1" valign="top"> Acr concentration(μM)</th></tr></thead><tbody><tr><td align="left" colspan="1" rowspan="1" valign="top">0.42</td><td align="left" colspan="1" rowspan="1" valign="top">48</td><td align="left" colspan="1" rowspan="1" valign="top">4.54 x 10<sup>14</sup></td><td align="left" colspan="1" rowspan="1" valign="top">3</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top">1</td><td align="left" colspan="1" rowspan="1" valign="top">55</td><td align="left" colspan="1" rowspan="1" valign="top">6 x10<sup>14</sup></td><td align="left" colspan="1" rowspan="1" valign="top">4</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top">1.68</td><td align="left" colspan="1" rowspan="1" valign="top">97</td><td align="left" colspan="1" rowspan="1" valign="top">1.81 x 10<sup>15</sup></td><td align="left" colspan="1" rowspan="1" valign="top">6</td></tr></tbody></table></table-wrap><table-wrap id="T5" position="float"><label>Table 5</label><caption><p> Molecular level interaction of A sample with citrate synthase. Assumption is molecular weights of Acr are 18 kDa and the substrate citrate synthase 85 kDa respectively.
The number of molecules of citrate synthase molecules has been calculated based on an assay volume of 0.30 ml and the number of citrate
synthase molecules is assumed to be 4.52 x 10<sup>14</sup>.</p></caption><table frame="hsides" rules="groups" width="100%"><thead><tr><th align="left" colspan="1" rowspan="1" valign="top">% citrate synthase covered</th><th align="left" colspan="1" rowspan="1" valign="top">%inhibition</th><th align="left" colspan="1" rowspan="1" valign="top">Molecules of Acr </th><th align="left" colspan="1" rowspan="1" valign="top"> Acr concentration (μM)</th></tr></thead><tbody><tr><td align="left" colspan="1" rowspan="1" valign="top">200</td><td align="left" colspan="1" rowspan="1" valign="top">95</td><td align="left" colspan="1" rowspan="1" valign="top">9.53 x 10<sup>14</sup></td><td align="left" colspan="1" rowspan="1" valign="top">5</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top"> 0</td><td align="left" colspan="1" rowspan="1" valign="top"> 0</td><td align="left" colspan="1" rowspan="1" valign="top"> 0</td><td align="left" colspan="1" rowspan="1" valign="top"> 0</td></tr></tbody></table></table-wrap></sec></sec><sec sec-type="Discussion" id="sec1-5"><title>5. Discussion</title><p>High levels of purified Acr were obtained after His- tag and gel filtration. Earlier studies have shown Acr to be a nonameric 158 kDa in the form of trimer of trimers, 210 kDa and 193 kDa ( <xref rid="ref4" ref-type="bibr">4</xref>, <xref rid="ref7" ref-type="bibr">7</xref>, <xref rid="ref8" ref-type="bibr">8</xref>, <xref rid="ref10" ref-type="bibr">10</xref>). The activity of A samples was two times less than that of B samples at 37°C. The difference in activity between A samples and B samples
could be explained by the difference in the ratio of oligomers in both the samples. The A samples showed 43% of 11 to 15 mers as compared
to the B samples which had 75% of 11 to 15 mers. In addition, the presence of 5 and 7 mers in the A samples was recorded. This could
explain the higher activity of the B samples. As the ratio of oligomers increases, the activity increases due to better molecular interaction
with the substrate. This is due to the physical coverage of the substrate and prevention of aggregation. In the case of citrate
synthase, as the molecular weight is larger, the mole ratio required is higher than insulin.</p><p>In the pre-heat treatment assays, it was conjectured that a more favourable secondary structure might aid in improving the chaperone
activity of gel-filtered Acr. Our observations indicate that pre-heating the sample might reduce the percentage of β sheets
and increase the randomness and disorderliness which corroborates with previous findings ( <xref rid="ref8" ref-type="bibr">8</xref>, <xref rid="ref10" ref-type="bibr">10</xref>, <xref rid="ref12" ref-type="bibr">12</xref>). However non-gel-filtered samples did not show any improvement suggesting an analysis of secondary structure that is required to
interpret this data. These results also suggest that more number of molecules of Acr can bind to insulin B chain and increase
in oligomer size of Acr enhances activity after pre-heat treatment.</p><p>In order to understand the molecular interactions preliminarily, a mole ratio of Acr to insulin versus percentage inhibition
was plotted (<xref ref-type="fig" rid="IJB-17-e2370-g003.tif">Figure 3a</xref>).
The assumption in the calculation of Acr activity is that the molecular weight is 18 kDa. However, the number of molecules would increase
if it is calculated in terms of an oligomer. In this regard, figures plotted are presented in terms of monomer and 9 mers in terms
of molecules of insulin covered and % inhibition (<xref ref-type="fig" rid="IJB-17-e2370-g003.tif">Fig. 3b, 3c</xref>). It has been reported from several previous studies about the occurrence
of Acr as a nonamer or dodecamer ( <xref rid="ref4" ref-type="bibr">4</xref>, <xref rid="ref7" ref-type="bibr">7</xref>). Our observation supports the previous findings that monomers aggregate to form oligomers that are able to bind effectively to inhibit insulin B chain aggregation. It also suggests that a higher ratio of oligomers would help binding insulin better. This is probably the basic mechanism by which heat shock proteins can prevent unfolding of proteins. The status of oligomerization of recombinant Acr protein affects chaperone activity against insulin B chain aggregation.Recent in silico studies on Acr have again assumed it to be a dodecamer ( <xref rid="ref15" ref-type="bibr">15</xref>).The potential of Acr as a diagnostic and vaccine is largely unfulfilled even 20 years after its discovery ( <xref rid="ref16" ref-type="bibr">16</xref>, <xref rid="ref17" ref-type="bibr">17</xref>).</p></sec><sec sec-type="Conclusions" id="sec1-6"><title>6. Conclusions</title><p>Soluble active Acr was purified by both Ni-NTA and the gel filtration chromatography to nearly 95% purity.
The chaperone activity of A samples (non-gel-filtered) against insulin at 37°C was 4 times less than that
of B samples (gel-filtered). The difference in activity could be explained by the difference in the ratio
of oligomers in both the samples as revealed by the Native-PAGE analysis. The activity of non-gel-filtered samples
(A) at 60°C showed 100% inhibition at a molar concentration of 44 µM which is higher than 24 µm achieved with non- gel-filtered
samples A at 37˚C.This difference in activity needs to be investigated by secondary structure analysis. To predict molecular
level interaction of Acr, polynomial graphs can be plotted from the preliminary activity data we obtained.
Molecular level interaction may be predicted in future for the in vivo condition in the host with the help of
these polynomial graphs. These could be useful tools to detect Acr protein inside the host and to verify our postulates.
Secondary structure analysis could be carried out to explain the correlation between structure and function.
Further verification can be carried out by <italic>in vivo</italic> isolation of <italic>M. tb</italic> Acr and studying its interactions
with insulin, citrate synthase and other substrates used <italic>in vitro</italic> to further strengthen the hypothesis.</p></sec></body><back><ack><title>Acknowledgement</title><p><italic>M. tb</italic> H37Rv genomic DNA was obtained from Dr. Mridula Bose, former Head of Department of Medical Microbiology, Patel Chest Institute, and New Delhi, India. Authors sincerely acknowledge the campus Director and Honorable Vice Chancellor of BITS Pilani University in the form of fellowship, visiting lecturership, and use of equipment, C- CAMP and Bugworks. We wish to thank Dr.Santanu Datta, Bugworks, GK’s co-guide and his colleagues for providing technical help in reviewing design of primers, troubleshooting expression issues and providing the Sephacryl S gel filtration 200 column and helping GK use their facilities.</p></ack><ref-list><title>References</title><ref id="ref1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Smith</surname><given-names>I</given-names></name></person-group><article-title> Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence</article-title><source> Clin Microbiol Rev</source><year> 2003</year><volume>16</volume><issue>3</issue><fpage>463</fpage><lpage>496</lpage><pub-id pub-id-type="doi">10.1128/cmr.16.3.463-496.2003</pub-id><pub-id pub-id-type="pmid">12857778</pub-id></element-citation></ref><ref id="ref2"><label>2</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kaufmann</surname><given-names>SH</given-names></name></person-group><article-title> Future vaccination strategies against tuberculosis: thinking outside the box</article-title><source> Immunity</source><year> 2010</year><volume>33</volume><issue>4</issue><fpage>567</fpage><lpage>577</lpage><pub-id pub-id-type="doi">10.1016/j.immuni.2010.09.015</pub-id><pub-id pub-id-type="pmid">21029966</pub-id></element-citation></ref><ref id="ref3"><label>3</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>De</surname><given-names>Jong W</given-names></name><name><surname>Leunissen</surname><given-names>J</given-names></name><name><surname>Voorter</surname><given-names>CEM</given-names></name></person-group><article-title> Evolution of the alpha-crystallin/small heat-shock protein family</article-title><source> Molecular Biology and Evolution</source><year> 1993</year><volume>10</volume><issue>1</issue><fpage>103</fpage><lpage>126</lpage><pub-id pub-id-type="doi">10.1093/oxfordjournals.molbev.a039992</pub-id><pub-id pub-id-type="pmid">8450753</pub-id></element-citation></ref><ref id="ref4"><label>4</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chang</surname><given-names>Z</given-names></name><name><surname>Primm</surname><given-names>TP</given-names></name><name><surname>Jakana</surname><given-names>J</given-names></name><name><surname>Lee</surname><given-names>IH</given-names></name><name><surname>Serysheva</surname><given-names>I</given-names></name><name><surname>Chiu</surname><given-names>W</given-names></name><etal></etal></person-group><article-title> Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3) functions as an oligomeric structure in vitro to suppress thermal aggregation</article-title><source>J Biol Chem</source><year> 1996</year><volume>271</volume><issue>12</issue><fpage>7218</fpage><lpage>7223</lpage><pub-id pub-id-type="pmid">8636160</pub-id></element-citation></ref><ref id="ref5"><label>5</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Valdez</surname><given-names>MM</given-names></name><name><surname>Clark</surname><given-names>JI</given-names></name><name><surname>Wu</surname><given-names>GJ</given-names></name><name><surname>Muchowski</surname><given-names>PJ</given-names></name></person-group><article-title> Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16</article-title><source>3 and human alphaB-crystallin</source><year> Eur J Biochem 2002</year><volume>269</volume><issue>7</issue><fpage>1806</fpage><lpage>1813</lpage><pub-id pub-id-type="doi">10.1046/j.1432-1033.2002.02812.x</pub-id><pub-id pub-id-type="pmid">11952782</pub-id></element-citation></ref><ref id="ref6"><label>6</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Preneta</surname><given-names>R</given-names></name><name><surname>Papavinasasundaram</surname><given-names>KG</given-names></name><name><surname>Cozzone</surname><given-names>AJ</given-names></name><name><surname>Duclos</surname><given-names>B</given-names></name></person-group><article-title> Autophosphorylation of the 16 kDa and 70 kDa antigens (Hsp 16.3 and Hsp 70) of Mycobacterium tuberculosis</article-title><source> Microbiology</source><year> 2004</year><volume>150</volume><issue>Pt 7</issue><fpage>2135</fpage><lpage>2141</lpage><pub-id pub-id-type="doi">10.1099/mic.0.26789-0</pub-id><pub-id pub-id-type="pmid">15256556</pub-id></element-citation></ref><ref id="ref7"><label>7</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kennaway</surname><given-names>CK</given-names></name><name><surname>Benesch</surname><given-names>JL</given-names></name><name><surname>Gohlke</surname><given-names>U</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Robinson</surname><given-names>CV</given-names></name><name><surname>Orlova</surname><given-names>EV</given-names></name><etal></etal></person-group><article-title> Dodecameric structure of the small heat shock protein Acr1 from Mycobacterium tuberculosis</article-title><source> J Biol Chem</source><year> 2005</year><volume>280</volume><issue>39</issue><fpage>33419</fpage><lpage>33425</lpage><pub-id pub-id-type="doi">10.1074/jbc.M504263200</pub-id><pub-id pub-id-type="pmid">16046399</pub-id></element-citation></ref><ref id="ref8"><label>8</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Panda</surname><given-names>AK</given-names></name><name><surname>Chakraborty</surname><given-names>A</given-names></name><name><surname>Nandi</surname><given-names>SK</given-names></name><name><surname>Kaushik</surname><given-names>A</given-names></name><name><surname>Biswas</surname><given-names>A</given-names></name></person-group><article-title> The C-terminal extension of Mycobacterium tuberculosis Hsp16.3 regulates its oligomerization, subunit exchange dynamics and chaperone function</article-title><source> FEBS J</source><year> 2017</year><volume>284</volume><issue>2</issue><fpage>277</fpage><lpage>300</lpage><pub-id pub-id-type="doi">10.1111/febs.13975</pub-id><pub-id pub-id-type="pmid">27885799</pub-id></element-citation></ref><ref id="ref9"><label>9</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname><given-names>H</given-names></name><name><surname>Huang</surname><given-names>S</given-names></name><name><surname>Dai</surname><given-names>H</given-names></name><name><surname>Gong</surname><given-names>Y</given-names></name><name><surname>Zheng</surname><given-names>C</given-names></name><name><surname>Chang</surname><given-names>Z</given-names></name></person-group><article-title>The Mycobacterium tuberculosis small heat shock protein Hsp16.3 exposes hydrophobic surfaces at mild conditions: conformational flexibility and molecular chaperone activi</article-title><source>Protein Sci</source><year> 1999</year><volume>8</volume><issue>1</issue><fpage>174</fpage><lpage>179</lpage><pub-id pub-id-type="doi">10.1110/ps.8.1.174PubMedPMID:10210195</pub-id><pub-id pub-id-type="pmid">10210195</pub-id></element-citation></ref><ref id="ref10"><label>10</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gu</surname><given-names>L</given-names></name><name><surname>Abulimiti</surname><given-names>A</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Chang</surname><given-names>Z</given-names></name></person-group><article-title> Monodisperse Hsp16.3 Nonamer Exhibits Dynamic Dissociation and Reassociation, with the Nonamer Dissociation Prerequisite for Chaperone-like Activity</article-title><source>J Mol Biol</source><year> 2002</year><volume>319</volume><issue>2</issue><fpage>517</fpage><lpage>526</lpage><pub-id pub-id-type="doi">10.1016/s0022-2836(02)00311-x</pub-id><pub-id pub-id-type="pmid">12051925</pub-id></element-citation></ref><ref id="ref11"><label>11</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Feng</surname><given-names>X</given-names></name><name><surname>Huang</surname><given-names>S</given-names></name><name><surname>Fu</surname><given-names>X</given-names></name><name><surname>Abulimiti</surname><given-names>A</given-names></name><name><surname>Chang</surname><given-names>Z</given-names></name></person-group><article-title> The reassembling process of the nonameric Mycobacterium tuberculosis small heat-shock protein Hsp16.3 occurs via a stepwise mechanism</article-title><source>Biochem J</source><year> 2002</year><volume>363</volume><issue>Pt 2</issue><fpage>329</fpage><lpage>334</lpage><pub-id pub-id-type="doi">10.1042/0264-6021:3630329</pub-id><pub-id pub-id-type="pmid">11931661</pub-id></element-citation></ref><ref id="ref12"><label>12</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fu</surname><given-names>X</given-names></name><name><surname>Jiao</surname><given-names>W</given-names></name><name><surname>Abulimiti</surname><given-names>A</given-names></name><name><surname>Chang</surname><given-names>Z</given-names></name></person-group><article-title> Inter-subunit cross- linking suppressed the dynamic oligomeric dissociation of Mycobacterium tuberculosis Hsp16.3 and reduced its chaperone activity</article-title><source>Biochemistry (Mosc)</source><year> 2004</year><volume>69</volume><issue>5</issue><fpage>552</fpage><lpage>557</lpage><pub-id pub-id-type="doi">10.1023/b:biry.0000029854.86015.61</pub-id><pub-id pub-id-type="pmid">15193130</pub-id></element-citation></ref><ref id="ref13"><label>13</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mao</surname><given-names>Q</given-names></name><name><surname>Ke</surname><given-names>D</given-names></name><name><surname>Feng</surname><given-names>X</given-names></name><name><surname>Chang</surname><given-names>Z</given-names></name></person-group><article-title> Preheat treatment for Mycobacterium tuberculosis Hsp16.3: correlation between a structural phase change at 60 degrees C and a dramatic increase in chaperone-like activity</article-title><source>Biochem Biophys Res Commun</source><year> 2001</year><volume>284</volume><issue>4</issue><fpage>942</fpage><lpage>947</lpage><pub-id pub-id-type="doi">10.1006/bbrc.2001.5074</pub-id><pub-id pub-id-type="pmid">11409884</pub-id></element-citation></ref><ref id="ref14"><label>14</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kurnellas</surname><given-names>MP</given-names></name><name><surname>Brownell</surname><given-names>SE</given-names></name><name><surname>Su</surname><given-names>L</given-names></name><name><surname>Malkovskiy</surname><given-names>AV</given-names></name><name><surname>Rajadas</surname><given-names>J</given-names></name><name><surname>Dolganov</surname><given-names>G</given-names></name><etal></etal></person-group><article-title> Chaperone activity of small heat shock proteins underlies therapeutic efficacy in experimental autoimmune encephalomyelitis</article-title><source> J Biol Chem</source><year>2012</year><volume>287</volume><issue>43</issue><fpage>36423</fpage><lpage>36434</lpage><pub-id pub-id-type="doi">10.1074/jbc.M112.371229</pub-id><pub-id pub-id-type="pmid">22955287</pub-id></element-citation></ref><ref id="ref15"><label>15</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Soong</surname><given-names>JX</given-names></name><name><surname>Lim</surname><given-names>TS</given-names></name><name><surname>Choong</surname><given-names>YS</given-names></name></person-group><article-title>The structural insights of 16.3 kDa heat shock protein (HSP16.3) from Mycobacterium tuberculosis via in silico study </article-title><source> Mol Simulation</source><year> 2017</year><volume>44</volume><issue>2</issue><fpage>117</fpage><lpage>127</lpage><pub-id pub-id-type="doi">10.1080/08927022.2017.1346254</pub-id></element-citation></ref><ref id="ref16"><label>16</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jee</surname><given-names>B</given-names></name><name><surname>Singh</surname><given-names>Y</given-names></name><name><surname>Yadav</surname><given-names>R</given-names></name><name><surname>Lang</surname><given-names>F</given-names></name></person-group><article-title> Small Heat Shock Protein16.3 of Mycobacterium tuberculosis: After Two Decades of Functional Characterization</article-title><source>Cell Physiol Biochem</source><year> 2018</year><volume>49</volume><issue>1</issue><fpage>368</fpage><lpage>380</lpage><pub-id pub-id-type="doi">10.1159/000492887</pub-id><pub-id pub-id-type="pmid">30138912</pub-id></element-citation></ref><ref id="ref17"><label>17</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Razavi</surname><given-names>SS</given-names></name><name><surname>Peyvandi</surname><given-names>H</given-names></name><name><surname>Badrkhani</surname><given-names>Jam AR</given-names></name><name><surname>Safari</surname><given-names>F</given-names></name><name><surname>Teymourian</surname><given-names>H</given-names></name><name><surname>Mohajerani</surname><given-names>SA</given-names></name></person-group><article-title> Magnesium Versus Bupivacaine Infiltration in Controlling Postoperative Pain in Inguinal Hernia Repair</article-title><source> Anesth Pain Med</source><year> 2015</year><volume>5</volume><issue>6</issue><fpage>e30643</fpage><pub-id pub-id-type="pmid">26705525</pub-id></element-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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