[Non-fused expression of HAb18GEF by reducing stability of translational initiation region in mRNA].
To express the extracellular fragement of hepatoma associated antigen HAbl8G(HAb18GEF) in E. coli efficiently in a non-fusing way, the cDNA of HAb18GEF gene was inserted into prokaryotic expression vector pET21a + . The secondary structure and codon adaptation of translational initiation region (TIR, from-30 to + 39) in mRNA of recombinant vector HAb18GEF/ pET21a + was predicted simultaneously by computer-aided design. Stable Stem-Loop structures and many low-usage codons were detected in mRNA-TIR of non-optimized recombinant HAb18GEF/pET21a + vector. The stability of mRNA-TIR in recombinant HAb18GEF/pET21a + vector was reduced with following methods: (1) optimization of secondary structure (2) optimization of codon adaptation. These optimization were realized by non-continual site-directed mutagenesis without changing any amino acid sequence in TIR. After being checked through restriction endonuclease digestion and confirmed through nucleotide sequencing, the pre-optimized and post-optimized recombinant vectors were transformed into competent E. coli JM109-DE3. The resulted recombinant clones were selected randomly and induced by IPTG at 37 degrees C. The induced production of these recombinants was analyzed by SDS-PAGE, indirect ELISA, Western blot, and cell fractionation assay. The amount of HAb18GEF mRNA was also detected by RNA dot blot between pre-optimized recombinant and post-optimized recombinant. The results revealed that recombinant non-fused vectors HAb18GEF/pET21a + were successfully constructed and optimized in the secondary structure and codon adaptation of TIR respectively. The HAb18GEF was expressed efficiently in a non-fusing way in recombinant E. coli by secondary structure optimization or codon adaptation optimization. Whereas, no expression of HAb18GEF was detected in pre-optimized recombinants. The non-fused expression products-HAb18GEF, mainly as inclusion body in E. coli, yielded highly above 29.3%. A trait of expression HAb18GEF was also detected both in intermembrane space and in culture medium due to over-expression and cell leakage. Difference in non-fused expression level of HAb18GEF between secondary structure optimization and codon adaptation optimization was negligible. No difference in amount of transcribed mRNA of HAb18GEF between the pre-optimized and the post-optimized recombinants was detected. To sum up, it's feasible to express hepatoma associated antigen HAb18GEF in a non-fusing way by reducing the stability of TIR in mRNA.
Zhang
Si-He
SH
Cell Engineering Research Center, Fourth Military Medical University, Xi' an 710032, China.
Xing
Jin-Liang
JL
Yao
Xi-Ying
XY
Chen
Zhi-Nan
ZN
D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D051926:Basigin / Q000096:biosynthesis* / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000235:genetics*; D020871:RNA Stability; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*
D000951:Antigens, Neoplasm; C497886:BSG protein, human; D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D051926:Basigin