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Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide

Paper ID Volume ID Publish Year Pages File Format Full-Text
23897 43481 2011 7 PDF Available
Title
Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide
Abstract

Five new expression vectors for recombinant human insulin production (pPT-B5Kpi, pPT-T10Rpi, pPT-T13Rpi, pPT-H27Rpi, pPT-B5Rpi), which have different sizes and leader peptide structure, were constructed and compared based on their expression level, yields of S-sulfonated preproinsulin (SSPPI) and folded proinsulin and enzymatic conversion rate. The ranking of expression level of the five fused proinsulins was H27R ≫ T10R > B5K > T13R ≈ B5R. In particular, the expression level of H27R was more than double (60–70%) the level of the other fused proinsulins, and this high expression level led to large amounts of SSPPI, folded proinsulin and insulin. Changes to the leader peptide structure affected not only protein expression level, but also refolding yield because the leader peptide affects protein conformation and hydrophobicity. The refolding yield of H27R was 85% at 500 L pilot scale. This high refolding yield was caused by the hydrophilic character of H27R. However, the β-mercaptoethanol concentration needed for refolding and the pH required to precipitate impurities after refolding had to be changed for high refolding yield. To avoid using CNBr, which is used to cleave fusion proteins, we used lysine and arginine linkers to connect the fusion protein and proinsulin. This fusion protein could be simultaneously cleaved by trypsin during enzymatic conversion to eliminate the C-peptide. The length and kind of leader peptide did not affect the enzyme reaction rate. Only the leader peptide linker connecting the B-chain influenced enzyme reaction rate. By testing several leader peptides, we constructed a new strain with 30% increased productivity based on expression level, refolding yield and enzyme reaction.

Keywords
Leader peptide; Sulfitolysis; S-sulfonated preproinsulin (SSPPI); Proinsulin; Enzymatic conversion
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Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 151, Issue 4, 20 February 2011, Pages 350–356
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