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Fragment complementation for the co-refolding of Thermotoga maritima β-glucosidase by gene splitting at non-homologous region

Paper ID Volume ID Publish Year Pages File Format Full-Text
18033 42710 2007 8 PDF Available
Title
Fragment complementation for the co-refolding of Thermotoga maritima β-glucosidase by gene splitting at non-homologous region
Abstract

β-Glucosidase from Thermotoga maritima is a 721 amino acid protein consisting of structurally distinct non-homologous region connecting the N- and C-terminal domains. To investigate the functional role of the non-homologous region in co-refolding, the gene was split at four sites (370, 403, 419 and 435) of the non-homologous region, cloned and separately expressed in E. coli generating eight peptide fragments (N370, N403, N419, N435, 371C, 404C, 420C and 436C). All eight fragments were recovered as insoluble inclusion bodies and found to be catalytically inactive. No catalytic activity was observed when these eight fragments were refolded individually. However, the catalytic activity was recovered when the two fragments derived from N- and C-terminal peptides were co-refolded together. Truncation of almost all amino acid residues in non-homologous region resulted in extremely low catalytic activity, which strongly suggests that non-homologous region is very important for the proper refolding of the peptides to reconstitute the catalytic activity. We succeeded in refolding the protein into an active form after splitting the gene at non-homologous region, denaturing and co-refolding the two domains. These results demonstrates the importance of structural elements that are not involved in the active site play an important role in protein folding to assemble the active site elements.

Keywords
β-Glucosidase; Thermotoga maritima; Non-homologous region; Co-refolding; Gene splitting; Inclusion bodies; Fragment complementation
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Fragment complementation for the co-refolding of Thermotoga maritima β-glucosidase by gene splitting at non-homologous region
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 4, 5 March 2007, Pages 732–739
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us