Biotechnology Based Process for Production of a Disulfide-Bridged Peptide.
A disulfide-bridged peptide drug development candidate contained two oligopeptide chains with 11 and 12 natural amino acids joined by a disulfide bond at the N-terminal end. An efficient biotechnology based process for the production of the disulfide-bridged peptide was developed. Initially, the two individual oligopeptide chains were prepared separately by designing different fusion proteins and expressing them in recombinant E. coli. Enzymatic or chemical cleavage of the two fusion proteins provided the two individual oligopeptide chains which could be conjugated via disulfide bond by conventional chemical reaction to the disulfide-bridged peptide. A novel heterodimeric system to bring the two oligopeptide chains closer and induce disulfide bond formation was designed by taking advantage of the self-assembly of a leucine zipper system. The heterodimeric approach involved designing fusion proteins with the acidic and basic components of the leucine zipper, additional amino acids to optimize interaction between the individual chains, specific cleavage sites, specific tag to ensure separation, and two individual oligopeptide chains. Computer modeling was used to identify the nature and number of amino acid residue to be inserted between the leucine zipper and oligopeptides for optimum interaction. Cloning and expression in rec E. coli, fermentation, followed by cell disruption resulted in the formation of heterodimeric protein with the interchain disulfide bond. Separation of the desired heterodimeric protein, followed by specific cleavage at methionine by cyanogen bromide provided the disulfide-bridged peptide.
Animesh
A
Goswami
Steven L
SL
Goldberg
Ronald L
RL
Hanson
Robert M
RM
Johnston
Olav K
OK
Lyngberg
Yeung
Y
Chan
Ehrlic
E
Lo
Steven H
SH
Chan
Nuria
N
de Mas
Antonio
A
Ramirez
Richard
R
Doyle
Wei
W
Ding
Discovery, R&D, Bristol-Myers Squibb , Route 206 and Province Line Road, Princeton, New Jersey 08543, United States.
Mian
M
Gao
Discovery, R&D, Bristol-Myers Squibb , Route 206 and Province Line Road, Princeton, New Jersey 08543, United States.
Stanley R
SR
Krystek
Discovery, R&D, Bristol-Myers Squibb , Route 206 and Province Line Road, Princeton, New Jersey 08543, United States.
Changhong
C
Wan
Discovery, R&D, Bristol-Myers Squibb , Route 206 and Province Line Road, Princeton, New Jersey 08543, United States.
Yeoun Jin
YJ
Kim
Discovery, R&D, Bristol-Myers Squibb , Route 206 and Province Line Road, Princeton, New Jersey 08543, United States.
Deepa
D
Calambur
Discovery, R&D, Bristol-Myers Squibb , Route 206 and Province Line Road, Princeton, New Jersey 08543, United States.
Mark
M
Witmer
Discovery, R&D, Bristol-Myers Squibb , Route 206 and Province Line Road, Princeton, New Jersey 08543, United States.
James W
JW
Bryson
D000595:Amino Acid Sequence; D001709:Biotechnology; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
D004220:Disulfides; D010455:Peptides