Cloning and expression of the human augmenter of liver regeneration at low temperature in Escherichia coli

doi:10.1016/j.jbbm.2006.11.009

Journal of Biochemical and Biophysical Methods

Volume 70, Issue 3, 10 April 2007, Pages 465-470

https://doi.org/10.1016/j.jbbm.2006.11.009 Get rights and content

Abstract

Acute and chronic hepatic failure is a devastating illness of varied causes with considerable mortality. Human augmenter of liver regeneration (hALR) is a hepatotrophic protein and the unique cytokine which can specially stimulate hepatic origin cells to grow regardless of genus. It has been proven that ALR can promote regeneration and avoid all kinds of injury in rat and canine models. In this study, the recombinant protein hALR was expressed successfully with recombinant prokaryotic expression vector pET28a(+) in Escherichia coli BL21 (DE3).

We constructed the recombinant expression vector pET28a(+)/hALR with a full-length cDNA encoding hALR protein from normal human liver tissue by one-step reverse transcription-polymerase chain reaction and his-tag recognition sequence encoding polyhistidine (6 × His). Under IPTG (isopropyl-β-d-thiogalactopyranoside) induction for 2 h at 37 °C, recombinant protein hALR was expressed. The expression of recombinant polyhistidine-tagged hALR was increased under low temperature and was confirmed that the temperature of 23 °C was the most suitable IPTG induction condition. Under low temperature induction of IPTG, recombinant protein can be expressed as a soluble protein. Recombinant protein hALR was also purified with His Bind Kits and characterized with SDS-PAGE and Western blotting.

The results showed that recombinant hALR could be expressed as a soluble protein under low temperature induction of IPTG. The successful expression of ALR in E. coli makes it possible to further study its biological function and purified recombinant hALR could be developed into a new anti-hepatic damage product.

Introduction

The liver has extraordinary regenerative power, and adequate regeneration usually occurs in patients with acute and chronic liver diseases, although extensive hepatocellular necrosis. However, in acute fulminant hepatic failure, the number of regenerative hepatocytes is obviously fewer than in surviving patients, and it appears that regeneration failure causes death in such patients [1], [2]. So, promoting hepatocellular regeneration is very important to improve the effects of conservation therapy [3], [4].

Recently, a novel hepatotrophic growth factor augmenter of liver regeneration (ALR), as described by its name, also called hepatic regenerative stimulator substance or hepatopoietin, has gained increased scientific interest [5], [6]. Since Higgins and Anderson discovered ALR in rats following 70% hepatectomy in 1931 [7], a series of research results has indicated that it increased the cell proliferative response following partial hepatectomy in the rat liver and was characterized as an important secondary hepatic growth factor. The latest research showed ALR is the only cytokine which can specially stimulate hepatic origin cells to proliferate regardless of genus and has been proved that ALR can significantly promote liver regeneration and avoid all kinds of injury in rat and canine models. Hagiya et al. [8] isolated a full-length cDNA clone encoding ALR factor from the cytosol of weanling rat including a 375 bp coding region. The encoded protein consists of 125 amino acids with a molecular weight of 15 kDa under reducing conditions. Giorda et al. [9] analyzed the genomic structure of the ALR gene in mouse and humans. Results showed that mouse and human ALR genes were found to be highly conserved and preferentially expressed in the testis and in the liver. Attempt to purified dog and human ALR also has been made by others [3]. The construction of recombinant expression vector pET28a(+)/hALR with a full-length cDNA encoding ALR protein from normal human liver tissue and his-tag recognition sequence encoding polyhistidine (6 × His) is included in our report. The recombinant polyhistidine-tagged hALR was expressed under the condition of low temperature induction in Escherichia coli BL21 (DE3) and purified using His-Bind column. We also characterized the recombinant polyhistidine-tagged hALR with SDS-PAGE and Western blotting.

Section snippets

Total RNA extraction

All experimental protocols and study methods were approved by the Animal Care Ethics Committee and Clinical Investigation Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University. Normal human liver tissue with 1 g obtained from a donor liver for orthotopic liver transplantation at the First Affiliated Hospital, College of Medicine, Zhejiang University. Informed consent was obtained from the relatives to utilize his liver for hALR mRNA extraction. Total cellular RNA

Obtained complete sequence of hALR cDNA by RT-PCR

The RT-PCR analysis revealed that the expected 423 bp of complete sequence hALR cDNA was obtained from normal human liver tissue. The RT-PCR product showed a single band of 423 bp on 1% agarose gel (Fig. 1A). DNA Sequencing confirmed that RT-PCR product encoding hALR protein was matched completely with hALR cDNA library in GeneBank (375 bp, GenBank Accession No: AF146394) and added sequences of BamH I restriction endonuclease, His-Tag recognition sequence, Factor Xa cleavage and Xho I

Discussion

The control of hepatic growth and regeneration has interested researchers for much of the last century [13], [14]. Since Higgins and Anderson [7] reported liver regeneration following a hepatectomy of 70% in rat in 1931, a good many of studies searching on growth factors within the liver have been performed [15], [16], [17], [18]. McJunkin and Breuhaus [19] described that the modest mitotic response to a hepatectomy of 30% to 40% in rats was enhanced with an intraperitoneal injection 2 days

Acknowledgments

This work was supported by grants from the: Natural Sciences Foundation of Zhejiang Province 2004B064, Science and Technology Foundation of Zhejiang Province 2006C23023, Zhejiang Health Science Foundation 2004B064, 2005A040 and 05Z008. We wish to thank Dr. Thomas Hui and John Guarino (Cedars-Sinai Medical Center, Los Angeles, CA 90048) for helpful comments and suggestions and critical reading of the manuscript.

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Cloning and expression of the human augmenter of liver regeneration at low temperature in Escherichia coli

Abstract

Introduction

Section snippets

Total RNA extraction

Obtained complete sequence of hALR cDNA by RT-PCR

Discussion

Acknowledgments

Dig Liver Dis

Dig Liver Dis

J Hepatol

Exp Cell Res

Steroids

Protein Expr Purif