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Acta Biochimica et Biophysica Sinica Advance Access originally published online on October 13, 2009
Acta Biochimica et Biophysica Sinica 2009 41(11):948-954; doi:10.1093/abbs/gmp089
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Expression and characterization of Kunitz domain 3 and C-terminal of human tissue factor pathway inhibitor-2

Lina Zhu1, Jiping Wang2, Jingui Mu2, Huijun Wang3, Chenqi Zhang1, Jue Wang1, Xingang Liu1, Xiaomin Yan1, Linsen Dai1,* and Duan Ma2,3,*

1 Center of Analysis and Measurement, Fudan University, Shanghai 200433, China
2 Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Fudan University, Shanghai 200032, China
3 Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China

* Correspondence address. Tel: +86-21-65643477; Fax: +86-21-65643014; E-mail: lsdai{at}fudan.edu.cn (L.D.); Tel: +86-21-54237441; Fax: +86-21-64033738; E-mail: duanma{at}shmu.edu.cn (D.M.)


  Abstract

Human tissue factor pathway inhibitor-2 (hTFPI-2) is a serine protease inhibitor and its inhibitory activity is enhanced by heparin. The Kunitz domain 3 and C-terminal of hTFPI-2 (hTFPI-2/KD3C), which has the activity toward heparin calcium, have been successfully expressed in Pichia pastoris and purified by SP-Sepharose and heparin-Sepharose chromatography. The Fourier transformed infrared spectroscopy (FTIR), Raman spectroscopy, and circular dichroism (CD) experiment results implied that hTFPI-2/KD3C contained small contents of {alpha}-helix and β-strand, but large amounts of random coil and two kinds of disulfide bonds, gauche-gauche-gauche (ggg) and trans-gauche-trans (tgt). The interaction of hTFPI-2/KD3C with heparin calcium was investigated by CD. It was found that heparin calcium induced β-strands in hTFPI-2/KD3C to different extents depending on the ratio of hTFPI-2/KD3C and heparin calcium.

Keywords    Kunitz domain 3 and C-terminal of hTFPI-2; heparin; secondary structure; Pichia pastoris

Received: April 28, 2009; Accepted: July 13, 2009
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