Non-fused expression of HAb18GEF by reducing stability of translational initiation region in mRNA.
- Author:
Si-He ZHANG
1
;
Jin-Liang XING
;
Xi-Ying YAO
;
Zhi-Nan CHEN
Author Information
1. Cell Engineering Research Center, Fourth Military Medical University, Xi' an 710032, China.
- Publication Type:Journal Article
- MeSH:
Antigens, Neoplasm;
biosynthesis;
genetics;
Base Sequence;
Basigin;
biosynthesis;
genetics;
Carcinoma, Hepatocellular;
genetics;
immunology;
Escherichia coli;
genetics;
metabolism;
Extracellular Matrix Proteins;
metabolism;
Genetic Vectors;
genetics;
Humans;
Liver Neoplasms;
genetics;
immunology;
Molecular Sequence Data;
Nucleic Acid Conformation;
Protein Biosynthesis;
genetics;
RNA Stability;
RNA, Messenger;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2004;20(2):175-180
- CountryChina
- Language:Chinese
-
Abstract:
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.
