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Production and purification of single chain human insulin precursors with various fusion peptides

  • Chung Woo Cho
  • Sun Ho Park
  • Doo Hyun Nam
Article

Abstract

For the production and purification of a single chain human insulin precursor four types of fusion peptides β-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)6-tagged sequence (HTS) were investigated. RecombinantE. coli harboring hybrid genes was cultivated at 37°C for 1 h, and gene induction occurred when 0.2 mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except forE. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0 mM IPTG, followed by a longer than 4 h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique

Keywords

fermentation fusion peptides gene induction purification single chain human insulin precursor 

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Copyright information

© The Korean Society for Biotechnology and Bioengineering 2001

Authors and Affiliations

  1. 1.Faculty of EngineeringKeimyung UniversityDaeguKorea
  2. 2.Biotechnology Research InstituteBicosys Co., Ltd.DaeguKorea
  3. 3.College of PharmacyYeungnam UniversityKyongsanKorea

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