Integrated production of human insulin and its C-peptide.
The potential for the development of an integrated process for production of human insulin and its C-peptide in Escherichia coli has been investigated. Human proinsulin was produced intracellularly in E. coli fused to two synthetic IgG-binding domains (ZZ) derived from staphylococcal protein A. High expression levels (3 g/l culture) of the gene product, which accumulated as inclusion bodies, was obtained. Solubilization of inclusion bodies by oxidative sulfitolysis and subsequent renaturation was performed directly after cell lysis and pellet wash. IgG affinity chromatography was used for efficient recovery of pure proinsulin fusion protein in a single step. Monomers of the proinsulin fusion protein constituted approximately 70%. A single step conversion of the fusion protein into insulin and C-peptide by trypsin and carboxypeptidase B treatment was achieved by engineering the junction between proinsulin and its affinity handle, ZZ. Characterization of the cleavage products by reversed phase chromatography (RPC) verified that human insulin and C-peptide were generated and that the ZZ affinity handle was resistant to cleavage. Human insulin and C-peptide were recovered with high yields by preparative reversed-phase high performance liquid chromatography (RP-HPLC). The potential use of the presented scheme for large-scale production of recombinant insulin and/or its C-peptide is discussed.
Nilsson
J
J
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Jonasson
P
P
Samuelsson
E
E
Ståhl
S
S
Uhlén
M
M
D001709:Biotechnology; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011384:Proinsulin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D007328:Insulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011384:Proinsulin