Expression, purification and evaluation of N-terminal domain of AcAP5 with Factor Xa inhibitory activity

doi:10.5246/jcps.2013.03.038

Abstract

Abstract:

Ancylostoma anticoagulant peptide 5 (AcAP5) is a strong inhibitor of human coagulation factor Xa (FXa). The N-terminal residues (N40) of AcAP5 contains a domain that could combine with FXa. In order to determine whether N40 protein has FXa inhibitory effect, we cloned, expressed and purified the protein for activity evaluation. The DNA fragment coding N40 was amplified by PCR, cloned into pET-30a to construct recombinant plasmid pET30a-N40, and subsequently transformed into E. coli. BL21 (DE3). Expression of N40 was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the interest protein was identified by SDS-PAGE and purified using one-step nickel (Ni) affinity chromatography. Under the optimal expression condition (0.05 mM IPTG for 6 h at 37 °C), the purity of N40 reached 90%. We also evaluated the inhibition activity of N40 protein on FXa, finding the IC50 was 4.58×10^-5mol/L. This study suggests the N40 of AcAP5 could combine with FXa to inhibit FXa activity.

Key words: AcAP5, FXa binding domain, FXa inhibition, Expression, Purification

CLC Number:

R962.1

Supporting:

Aihua Liu, Yuanjun Zhu, Xiaoyan Liu, Yinye Wang^*. Expression, purification and evaluation of N-terminal domain of AcAP5 with Factor Xa inhibitory activity[J]. , DOI: 10.5246/jcps.2013.03.038.

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