Christopher A. Dadd

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.

CHAPTER 17 – Nonradioactive Sequencing of Random Peptide Recombinant Phage

This chapter describes nonradioactive sequencing of random peptide recombinant phage. Issues of cost, disposal, and safety have made nonradioactive detection methods for DNA sequencing and other probe hybridization techniques as the method of choice for many laboratories. Sanger di-deoxynucleotide chain-termination sequencing with a biotinylated oligonucleotide primer and subsequent chemiluminescent detection results in autoradiograms that rival the sensitivity of standard 32p and 3SS sequencing methodology. The standard protocol consists of three major parts that include asymmetric amplification of single-stranded phage DNA, cycle sequencing with di-deoxynucleotides and a biotinylated primer, and electroblotting of the sequencing gel onto a nylon membrane with detection of the transferred DNA by chemiluminescence. It is found that when using the replication-defective fUSE phage, it is necessary to amplify the phage from a single transfected colony to obtain enough single-stranded phage DNA for sequencing. It is observed that chemiluminescent detection of sequencing fragments requires transfer of the biotinylated DNA to a positively charged nylon-66 membrane, such as Tropilon Plus.