Production and functional characterization of a recombinant fragment of von Willebrand factor (vWF): an antagonist to platelet receptor GP Ib.
We expressed a recombinant peptide fragment (Ser445-Val733) of human von Willebrand factor (vWF), containing the binding domain for the platelet receptor of GP Ib, in E. coli. This 33 kD peptide blocks binding of the intact vWF molecule to GP Ib in the presence of modulators. Thus, it offers potential as an antithrombotic agent. High level expression was achieved in a plasmid construct driven by the bacteriophage T7 promoter. The peptide was solubilized from inclusion bodies in strong chaotrope, then reduced and alkylated. Following purification, formulation at pH 3.5, and lyophilization, the reconstituted experimental product (RG 12986) exists as an equilibrium of monomer and dimer species. When formulated above pH 5.0, soluble aggregates are formed; these solutions have less bioactivity than RG 12986. Interestingly, the non-aggregated state of RG 12986 remains conserved following dilution and incubation with platelet-poor plasma. The overall purification/low pH formulation strategies may be applicable to other E. coli recombinant proteins having a tendency to aggregate following removal of chaotrope near physiologic pH when in a concentrated format.
Biotechnology Division, Phône-Poulenc Rorer, King of Prussia, PA 19406.
C
C
Prior
V
V
Chu
J
J
Holt
V
V
Windisch
T
T
Lee
J
J
Mitschelen
J
J
Newman
G
G
Ricca
C
C
Tarr
M
M
Hrinda
D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D010446:Peptide Fragments / Q000302:isolation & purification / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects*; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D014841:von Willebrand Factor / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology
D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor