Purification of a bioactive recombinant human Reg IV expressed in Escherichia coli
Introduction
Regenerating gene (Reg) IV1 is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily and was firstly isolated from a cDNA library of ulcerative colitis (UC) tissues by Hartupee et al. [1]. Human Reg IV is synthesized as a 158 amino acid (aa) precursor with a 22 aa signal sequence and a 136 aa mature chain [2]. The molecular weight (MW) of mature Reg IV protein is about 16 kDa [3]. Amino acids 37–155 constitute a C-type lectin-like domain, and aa residue 50 is a site for potential N-linked glycosylation. In addition, aa residue 127 and 142–143 form a ligand-binding surface (SwissProt # Q9BYZ8). Human Reg IV shares 68% and 67% aa sequence identity with rat and mouse Reg IV, respectively. Reg IV is weakly expressed in normal colon and stomach, and strongly expressed in colon adenocarcinoma [4], [5], pancreatic cancer [6], gastric adenocarcinoma [7], [8], [9], appendiceal mucinous cystadenoma and pseudomyxoma peritonei [10], inflammatory bowel disease (Crohn’s disease and ulcerative colitis) [1], [3], [11], and outside the gastrointestinal tract in prostate cancer [12], [13]. The physiological function of Reg IV and the functional signaling pathway(s) activated by Reg IV are presently unknown, but it may be involved in regeneration, cell growth and survival, cell adhesion, and resistance to apoptosis [9].
Large quantity of highly purified recombinant human Reg IV (rhReg IV) protein is needed to delineate the role of Reg IV in vitro and especially in vivo. Although human Reg IV has been purified previously using the Pichia pastoris expression system [14], two additional amino acids (Glu–Phe) were added to the N-terminus of mature Reg IV and two forms of the protein existed in the final preparation. However, the Escherichia coli (E. coli) derived rhReg IV is commercially available, demonstrating the success of E. coli expression system in making the protein. But no reference and protocol are disclosed from any of the vendors selling the protein and the high price and unpublished method prevent its application in animal studies which require the protein in milligram quantity. We therefore developed a new method of making the protein in milligram scale with high quality to meet the demand of animal studies.
In this report, we describe a new method for making rhReg IV using the E. coli expression system. The protein expressed in bacteria inclusion bodies was denatured and refolded in defined conditions, followed by one-step ion-exchange chromatography purification. The biological activity of the purified protein was determined by the reported methods of HCT116 and HT29 cell proliferation assays [15]. This simple protocol yields milligram quantities of protein, and is the first example of high level Reg IV purification from a prokaryotic expression system.
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Vectors, strains, and supplies
The expression vector pET30a (+) and host strain E. coli DH5α and BL21 (DE3) were purchased from Novagen (Germany). Homo sapiens regenerating gene family, member 4, mRNA (cDNA clone), complete cds (coding sequence) was purchased from GeneCopoeia (USA). Restriction enzymes were supplied by Fermentas (USA). DNA polymerase (KOD plus) was purchased from Toyoba Company (Japan). Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Merck (Germany). All other chemicals and reagents were from
Expression of rhReg IV
E. coli harboring pET30a–rhReg IV were cultured in LB medium at 37 °C and IPTG was added when the culture reached the OD600 of 0.6–0.8 to induce expression of rhReg IV. The expression of ∼16 kDa protein was induced as revealed by Coomassie brilliant blue R-250 staining of the protein gel separated by SDS–PAGE (Fig. 2a, Lane 2). The identity of the expressed protein was investigated by Western blotting using commercial available anti-Reg IV antibody (Santa Cruz, USA). The un-induced and induced E.
Discussion
In the present study, we have developed a simple protocol for purification of Reg IV using a bacterial expression system without the use of protein tags such as GST or Histidine. By overexpressing the protein in E. coli, isolating inclusion bodies, and refolding the proteins to its native conformation, we obtained milligram quantities of Reg IV. Initial attempts to refold the protein using an approach similar to that used previously in our laboratory to refold the mouse Mig expressed in
Acknowledgment
This work was supported in part by the China Ministry of Science and Technology (grant “863” 2007AA02Z149; “New drug initiative” 2009ZX09103-743 to W.H.) and The Science and Technology Commission of Shanghai Municipality (grant 075407071 to W.H.).
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