[Expression of a human myofibrillogenesis regulator 1 gene in E. coli and its immunogenicity].
OBJECTIVE
To study the expression of human myofibrillogenesis regulator 1 (MR-1) gene in E. coli and obtain the MR-1 protein and its antibody for further investigation of its biological function.


METHODS
Expression vectors pGEX-5X-1, pET30a (+), and pET24a (+), as well as host strain E. coli BL21 (DE3) and BL21-CodonPlus (DE3) -RIL were used for expression of MR-1. MR-1 N-terminal with GST or T7-tag or C-terminal with His-tag, separately, or N terminal with T7-tag and C terminal with His-tag, simultaneously, were fused in plasmids pGEX-5X-1, pET30a (+) , and pET24a (+). The expressed MR-1-T protein, separated and purified by preparative SDS-PAGE, was applied to immunize the rabbits. The titer of the antibody was assayed by ELISA and its immunogenicity was tested by Western blot with pcDNA3/MR-1 transfected human breast cancer cell MCF7.


RESULTS
The MR-1 protein was successfully expressed as inclusion body by fusing its N-terminal with T7-tag in E. coli BL21-CodonPlus (DE3) -RIL. MR-1 protein was purified by electro-elution from SDS-PAGE gel. Using this purified protein, polyclonal antibody in rabbit against MR-1 was essentially generated. ELISA and Western blot showed the titer of this antibody was about 1:10(5) with high immunogenicity.


CONCLUSIONS
The N-terminal fusion tag is the most important mechanism for MR-1 expression. The polyclonal antibody of the generated MR-1 protein in E. coli may be applied for its further biological function studies.
Li
Tian-bo
TB
Department of Microbial Pathway Engineering, Institute of Medicinal Biotechnology, CAMS and PUMC, Beijing 100050, China.
Hu
Yang
Y
Feng
Shuang
S
Zuo
Zeng-yan
ZY
Wang
Yi-guang
YG
D000818:Animals; D000906:Antibodies / Q000032:analysis; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000378:metabolism*; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D007114:Immunization; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014162:Transfection
D000906:Antibodies; D009124:Muscle Proteins; C495444:PNKD protein, human; D011993:Recombinant Fusion Proteins