Human insulin production from a novel mini-proinsulin which has high receptor-binding activity.
To increase the folding efficiency of the insulin precursor and the production yield of insulin, we have designed a mini-proinsulin (M2PI) having the central C-peptide region replaced with a sequence forming a reverse turn. The mini-proinsulin was fused at the N-terminus to a 21-residue fusion partner containing a His10 tag for affinity purification. The gene for the fusion protein was inserted downstream of the T7 promoter of the expression plasmid pET-3a, and the fusion proteins were produced as inclusion bodies in the Escherichia coli cytoplasm at levels up to 25% of the total cell protein. The protein was sulphonated, cleaved by CNBr and the M2PI mini-proinsulin was purified using ion-exchange chromatography. The refolding yield of M2PI was 20-40% better than that of proinsulin studied at the same molar concentrations, indicating that the short turn-forming sequence is more effective in the refolding process than the much longer C-peptide. Native human insulin was successfully generated by subsequent enzymic conversion of mini-proinsulin. The mini-proinsulin exhibited high receptor-binding activity, about 50% as potent as insulin, suggesting that this single-chained mini-proinsulin may provide a foundation in understanding the receptor-bound structure of insulin as well as the role of C-peptide in the folding and activity of proinsulin.
Chang
S G
SG
Laboratory of Protein Engineering and Molecular Design, Hanhyo Institute of Technology, Taejon, Korea.
Kim
D Y
DY
Choi
K D
KD
Shin
J M
JM
Shin
H C
HC
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002460:Cell Line; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000096:biosynthesis*; D008214:Lymphocytes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017510:Protein Folding; D011972:Receptor, Insulin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000138:chemical synthesis
D007328:Insulin; D011993:Recombinant Fusion Proteins; C060793:miniproinsulin; D011384:Proinsulin; D011972:Receptor, Insulin