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 500L 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.
authors.affiliation: Dept. of Integrated Biotechnology, Sogang University, Seoul 121-742, Republic of Korea.
authors.forename: Cheol-Ki
authors.initials: CK
authors.lastname: Min
authors.forename: Young-Jin
authors.initials: YJ
authors.lastname: Son
authors.forename: Chang-Kyu
authors.initials: CK
authors.lastname: Kim
authors.forename: Sang-Joong
authors.initials: SJ
authors.lastname: Park
authors.forename: Jin-Won
authors.initials: JW
authors.lastname: Lee
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D011384:Proinsulin / Q000737:chemistry; D011487:Protein Conformation; D011498:Protein Precursors / Q000032:analysis / Q000737:chemistry; D058849:Protein Refolding; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics*; D014357:Trypsin / Q000737:chemistry
chemical_list: D007328:Insulin; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C013737:preproinsulin; D011384:Proinsulin; D014357:Trypsin