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World J Gastroenterol. Jul 21, 2006; 12(27): 4401-4405
Published online Jul 21, 2006. doi: 10.3748/wjg.v12.i27.4401
Expression, purification and bioactivity of human augmenter of liver regeneration
Yang-De Zhang, Jian Zhou, Jin-Feng Zhao, Jian Peng, Xiao-Dong Liu, Xin-Sheng Liu, Ze-Ming Jia
Yang-De Zhang, Jin-Feng Zhao, Xiao-Dong Liu, National Hepatobiliary and Enteric Surgery Research Center, Ministry of Health, Changsha 410008, Hunan Province, China
Jian Zhou, Jian Peng, Xin-Sheng Liu, Ze-Ming Jia, Xiangya Hospital of Central South University, Changsha 410008, Hunan Province, China
Supported by National “863” Program of China , No. 2002AA214011
Correspondence to: Professor Yang-De Zhang, National Hepatobiliary and Enteric Surgery Research Center, Ministry of Health, 141 Xiangya Road, Changsha 410008, Hunan Province, China. zyd@2118.cn
Telephone: +86-731-4327939 Fax: +86-731-4327987
Received: March 11, 2004
Revised: April 28, 2004
Accepted: August 3, 2004
Published online: July 21, 2006
Abstract

AIM: To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration (hALR) and to study their biological activity.

METHODS: hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase (GST) sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation.

RESULTS: The product of PCR from plasmid pGEM-T-hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups (P < 0.01).

CONCLUSION: Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application.

Keywords: Human augmenter of liver regeneration; Gene recombination; Expression; Purification; Fusion protein; Transformation; Biological activity