[On-column refolding and purification of human EGF receptor L2 domain inclusion body overexpressed in Escherichia coli].
The human epidermal growth factor receptor (EGFR) extracellular region (residues 1-621) consists of four subdomains, i.e. L1, S1, L2, and S2. The L2 domain (EGFR-L2) is composed of residues 311-479 and plays a major role in ligand-binding. Due to the high content of cysteine residues (42 cysteines) in the S1 and S2 domains, it is quite difficulty to get a correctly refolded product of the complete EGFR extracellular domain. In contrast, only 4 cysteine residues are present in EGFR-L2 domain. The aim of the present study is to prepare a soluble EGFR-L2 domain from the recombinant protein inclusion body overexpressed in Escherichia coli (E. coli). DNA fragment encoding EGFR-L2 containing a polyhistidine-tag at the carboxyl terminus was amplified by PCR from the cDNA of EGFR extracellular region, and was inserted into pET-3c to construct the prokaryotic expression vector. The target protein was highly expressed in E. coli BL21 (DE3) strain and was only present in the inclusion body as revealed by immunoblotting analysis. No soluble product could be refolded through dilution or stepwise dialysis strategies. However, on-column refolding of denatured EGFR-L2 bound to Ni2+ -NTA produced a soluble one. Furthermore,the soluble EGFR-L2 was simultaneously purified to high purity (>95%) through eluting from the same Ni2+ -NTA column with a linear imidazole gradient. The refolded EGFR-L2 had specific binding activity with the cognate ligand EGF, although its affinity was low. These results suggest that a polyhistidine-tag fused with a recombinant protein facilitate not only the purification but also the renaturation of the target product through on-column refolding. Besides, this refolding strategy may be suitable for the preparation of those recombinant proteins which are hard to refold through conventional approaches.
Xu
Li-Hui
LH
Key Laboratory of Ministry of Education for Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China.
Hong
An
A
He
Xian-Hui
XH
D066246:ErbB Receptors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors