Role of Molecular Interactions and Oligomerization in Chaperone Activity of Recombinant Acr from Mycobacterium tuberculosis
Identifieur interne : 000399 ( Main/Exploration ); précédent : 000398; suivant : 000400Role of Molecular Interactions and Oligomerization in Chaperone Activity of Recombinant Acr from Mycobacterium tuberculosis
Auteurs : Gautam Krishnan [Inde] ; Utpal Roy [Inde]Source :
- 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
Affiliations:
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<author><name sortKey="Krishnan, Gautam" sort="Krishnan, Gautam" uniqKey="Krishnan G" first="Gautam" last="Krishnan">Gautam Krishnan</name>
<affiliation wicri:level="1"><nlm:aff id="aff1"> Department of Biological Sciences, BITS Pilani KK Birla Goa Campus, Zuari Nagar, Goa 403726, Goa, India</nlm:aff>
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<author><name sortKey="Roy, Utpal" sort="Roy, Utpal" uniqKey="Roy U" first="Utpal" last="Roy">Utpal Roy</name>
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<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>
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<author><name sortKey="Krishnan, Gautam" sort="Krishnan, Gautam" uniqKey="Krishnan G" first="Gautam" last="Krishnan">Gautam Krishnan</name>
<affiliation wicri:level="1"><nlm:aff id="aff1"> Department of Biological Sciences, BITS Pilani KK Birla Goa Campus, Zuari Nagar, Goa 403726, Goa, India</nlm:aff>
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<author><name sortKey="Roy, Utpal" sort="Roy, Utpal" uniqKey="Roy U" first="Utpal" last="Roy">Utpal Roy</name>
<affiliation wicri:level="1"><nlm:aff id="aff1"> Department of Biological Sciences, BITS Pilani KK Birla Goa Campus, Zuari Nagar, Goa 403726, Goa, India</nlm:aff>
<country xml:lang="fr">Inde</country>
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<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>
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<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>
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