Identification and characterization of protease inhibition effect of Kunitz-type serine protease inhibitor 1(NaKuI1)from Necator americanus

Abstract

Abstract: Objective To isolate and express a Kunitz-type serine protease inhibitor, named NaKuI1, from the human hookworm Necator americanus, and identify its protease inhibitory activity. Methods Primers were designed based on the predicted nucleotide sequence GenBank No.XM_013449790, which coded for a predicted Kunitz type serine protease inhibitor consisting of 63-amino acids. The 3′ and 5′ ends of NaKuI1 cDNA were amplified from the N. americanus adult worm cDNA with rapid amplification of cDNA ends (SMART-RACE) technique. The nucleotide sequence encoding mature NaKuI1 was cloned and ligated into pET32a-sumo vector to construct the recombinant plasmid pET32a-sumo/NaKuI1, which was then transferred into Escherichia coli BL21 (DE3). Expression of NaKuI1 fusion protein was induced with IPTG. The expressed products in inclusion bodies were denatured, refolded, then purified by Ni-NTA resin affinity chromatography, and cleaved with SUMO protease to obtain the recombinant protein rNaKuI1. Activated partial thromboplastin time (aPTT) and prothrombin time (PT) assays were used to explore the anticoagulant activity of rNaKuI1, and a single-stage chromogenic assay was used to detect the inhibitory effect on serine proteases. Results The full-length cDNA of NaKuI1 was obtained, and it includes an open-reading frame of 255 nucleotides encoding a 84-amino-acid peptide, consisting of a signal peptide of 16 amino acids and a mature peptide of 68 amino acids. NaKuI1 fusion protein expressed in E. coli BL21 (DE3) was in a form of inclusion body, and purified rNaKuI1 had no anticoagulant activity. At a 100-fold molar ratio, rNaKuI1 exhibited nearly 100% inhibition on enzymatic activity of human fibrinolytic enzyme (5 nmol/L), human trypsin (1 nmol/L) and porcine trypsin (5 nmol/L), and 31.45% and 25.18% inhibition on bovine pancreatic α-chymotrypsin (1 nmol/L) and human neutrophil elastase (5 nmol/L), respectively. However, it did not affect the enzymatic activity of human cathepsin G, proteinase 3 and porcine pancreatic elastase. rNaKuI1 inhibited human pancreatic trypsin and plasmin with ki values of(21.17 ± 7.22) nmol/L and(21.72 ± 3.95) nmol/L, respectively. Conclusion The full-length cDNA of NaKuI1 is obtained, which shows strong inhibitory activities against trypsin and plasmin.

Key words: Necator americanus, Kunitz-type serine protease, Inhibitor, Prokaryotic expression, Trysin, Plasmin

CLC Number:

R383.132

Xi-xiang SUI, Zheng SHAO, Qi JIANG, Ye ZHOU, Qing-feng HE, Yong-li SITU, Li DENG, Li-fei PENG. Identification and characterization of protease inhibition effect of Kunitz-type serine protease inhibitor 1(NaKuI1)from Necator americanus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2017, 35(3): 259-264.

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