Purification and refolding of a novel cancer/testis antigen BJ-HCC-2 expressed in the inclusion bodies of Escherichia coli.
BJ-HCC-2 is one of the cancer/testis antigens that may be the most promising targets for tumor immunotherapy. To investigate the expression of BJ-HCC-2 protein in tumor cells and its capacity to elicit CTL response, the recombinant protein of BJ-HCC-2 was expressed in the inclusion bodies in Escherichia coli. The inclusion bodies were solubilized effectively with 0.3% N-lauroyl sarcosine in alkaline buffer. Under this denatured form, the BJ-HCC-2 protein carrying 6x histidine tag was purified with Ni-NTA affinity chromatography in a single step with a purity of over 97%. The yield of the purified protein was about 78%. The purified recombinant protein was refolded in a simple way. The correct refolding of the recombinant protein was verified in the recovery of its secondary and tertiary structures as assessed by circular dichroism and fluorescence emission spectra. The recovery rate of refolded protein was 92.1%. The renatured protein displayed its immunoreactivity with the antibodies to BJ-HCC-2 protein by Western blotting. This method of protein purification and refolding is easy to manipulate and may be applicable to the hydrophobic proteins that are unable to be purified by other methods.
Yang
Xiao Ang
XA
Department of Immunology, School of Basic Medical Science, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100083, China. xiaoangyang@hotmail.com
Dong
Xue Yuan
XY
Li
Yan
Y
Wang
Yue Dan
YD
Chen
Wei Feng
WF
D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D010957:Plasmids; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013736:Testicular Neoplasms / Q000276:immunology / Q000473:pathology; D014157:Transcription Factors
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; C449758:FATE1 protein, human; D011994:Recombinant Proteins; D014157:Transcription Factors