Affinity purification of von Willebrand factor using ligands derived from peptide libraries.
The chromatographic purification of vWF (von Willebrand Factor) from human plasma represents a challenge because it consists of multimers with molecular weights ranging from 0.5 to 10 million Daltons. Phage peptide library screening yielded a lead peptide (RLRSFY) that interacts with vWF. Conservative substitutions of terminal residues of the lead peptide led to a second peptide, RVRSFY, which was more efficient in the affinity chromatographic purification of vWF from protein mixtures. Adsorption isotherm measurements indicated multiple interactions between vWF and the immobilized peptide RVRSFY. Increases in peptide density on the chromatographic supports resulted in stronger association constants and higher maximum protein binding capacities. When the peptide density was lower than 32 mg/mL, there was no measurable interaction between vWF and immobilized peptide RVRSFY in HEPES buffer containing 0.5 M NaCl at pH 7. An increase in peptide density from 32 to 60 mg/mL increased the association constants from 0.9 x 10(6) to 2 x 10(6) (M-1). Divalent salts (calcium and magnesium chloride) were used to elute the retained vWF with 82.5% of the activity recovered. The interactions between vWF and the immobilized peptide RVRSFY are dominated by ionic attractions and also involve hydrophobic interactions at close contact. Finally, the purification of vWF from crude material PEG filtrate of a cryoprecipitate of human plasma is demonstrated using affinity chromatography with immobilized N-acetyl-RVRSFYK.
Huang
P Y
PY
Department of Chemical Engineering, North Carolina State University, Raleigh 27695-7905, USA.
Baumbach
G A
GA
Dadd
C A
CA
Buettner
J A
JA
Masecar
B L
BL
Hentsch
M
M
Hammond
D J
DJ
Carbonell
R G
RG
D000327:Adsorption; D000595:Amino Acid Sequence; D000818:Animals; D002021:Buffers; D002413:Cations, Divalent / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D009994:Osmolar Concentration; D011817:Rabbits; D013499:Surface Properties; D013696:Temperature; D014841:von Willebrand Factor / Q000302:isolation & purification* / Q000378:metabolism
D002021:Buffers; D002413:Cations, Divalent; D008024:Ligands; D009842:Oligopeptides; D014841:von Willebrand Factor