Gene modification and high prokaryotic expression of porcine interferon alpha-1.
- Author:
Rui-Bing CAO
1
;
Xue-Qing XU
;
Bin ZHOU
;
De-Sheng CHEN
;
Pu-Yan CHEN
Author Information
1. Key Laboratory of Animal Disease Diagnostic and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Codon;
genetics;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
Interferon-alpha;
biosynthesis;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
Swine;
Transduction, Genetic
- From:
Chinese Journal of Biotechnology
2004;20(2):291-294
- CountryChina
- Language:Chinese
-
Abstract:
There are many E. coli rare codons in the gene of porcine interferon alpha-1. In order to obtain high expression of poIFN-alpha1 in E. coli, the cDNA encoded poIFN-alpha1 mature protein was synthesized using biased codons of E. coli without changing the original amino acid sequence and the terminator was changed as TAA. At the same time, Adenine and Thymine were used to the largest extent near the 5' terminus of poIFN-alpha1 mature protein gene. The synthesized gene was inserted into the Eco RI and Sal I site of the expression vector pRLC resulting pRLC-poIFN-alpha1. The poIFN-alpha1 is highly expressed in E. coli DH5alpha when the induction was carried out at 42 degrees C . The expressed poIFN-alpha1 account for 24.5% of the total cellular proteins and existed as inclusion body. The poIFN-alpha1 inclusion body was dissolved in 6mol/L guanidine chloride contained DTT and subsequently the denatured poIFN-alpha1 was re-natured by dilution in refolding buffer containing GSH and GSSH. In the present study it was found that the denatured poIFN-alpha1 was most efficiently re-natured in refolding buffer containing 1 mol/L guanidine chloride. In order to obtain pure protein, the concentrated re-natured poIFN-alpha1 was purified by Sephacryl S-200 chromatography. As a result, the purified poIFN-alpha1 is verified to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in MDBK cells, which is about 6.4 x 10(6) u/mg. This study paved the way for large-scale production of recombinant poIFN-alpha1 and its usage in virus disease control of pigs.
