Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins.

We previously reported the purification of a serine proteinase from the venom of the sharp-nosed pit viper (Deinagkistrodon acutus) using a combination of affinity chromatography and ion-exchange chromatography [Xin, Dong, Wang and Li, R. (2007) J. Chromatogr. B 859, 111-118]. The high fibrinogen-clotting activity [2025 NIH (National Institutes of Health) units/mg] of this protein indicated that it may have great potential as a drug for treating thrombolysis. In order to systemically determine the purified protein's structure and activity, it was characterized using the following methods: MS, isoelectric focusing, deglycosylation analysis, amino acid composition analysis, peptide mass fingerprinting, N-terminal amino acid sequencing, CD, hydrophobic-site analysis and bioactivity assays. In addition, a fluorescence probe was synthesized and conjugated to the protein in order to analyse its active site. The results indicated that the protein is a novel acutobin-like enzyme (designated acutobin II) with strong clotting and esterase activities and is composed of a 28 kDa peptide chain plus approx. 6 kDa of O-linked glycan chains. The protein contains 249 amino acids and, remarkably, no tryptophan residues. The pI of the protein is 4.8+/-0.2. The protein's secondary structure is dominated by beta-sheets (49%) and random coils (43%), and its tertiary structure does not contain any metal ions or disulfide bonds and possesses only one hydrophobic pocket. Analysis revealed that the hydrophobic pocket is most likely the enzymatic active site.