(A-C-B) human proinsulin, a novel insulin agonist and intermediate in the synthesis of biosynthetic human insulin.
The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide. Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin. A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain. To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free. Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein. By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed-phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity. Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding. Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin. The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin. Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation. The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin.
Heath
W F
WF
Lilly Research Laboratories, Indianapolis, Indiana 46285.
Belagaje
R M
RM
Brooke
G S
GS
Chance
R E
RE
Hoffmann
J A
JA
Long
H B
HB
Reams
S G
SG
Roundtree
C
C
Shaw
W N
WN
Slieker
L J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D007003:Hypoglycemia / Q000139:chemically induced; D007328:Insulin / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010920:Placenta / Q000378:metabolism; D010957:Plasmids; D011384:Proinsulin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014170:Transformation, Genetic
D007328:Insulin; D013438:Sulfhydryl Compounds; C072204:(A-C-B) human proinsulin; D011384:Proinsulin; D011972:Receptor, Insulin