Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology  2017, Vol. 37 Issue (1): 14-20    DOI: 10.13523/j.cb.20170103
    
Comparison of Expression and Activity of Antiviral Protein RC28 in Escherichia coli and Pichia pastoris
HU Li-qiang, ZHENG Wen, ZHONG Yi, DU Dan, YANG Hao, GONG Meng
HuaXi-Washington Mitochondria and Metabolism Research Center, West China Hospital, SCU, Chengdu 610041, China
Download: HTML   PDF(635KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

RC28 is a novel antiviral protein extracted from the mushroom Rozites caperata. In our previous studies, RC28 cDNA were obtained from Rozites caperata total RNA by 3'-RACE and inserted into pMD18-T plasmid by TA cloning. RC28 cDNA fragments were sub-cloned into E. coli expression vector pET28a(+) and Pichia yeast expression vector pPIC9K separately to form recombinant RC28 expression vectors with N'-terminal histidine tags. The two kinds of RC28 expression vectors were transformed into their respective host strains and positive clones were selected to construct stable expression systems. SDA-PAGE and Western blot were performed to analyze RC28 expression after induction. RC28 protein were purified and collected by using Ni-NTA columns and detection of the antiviral activity of the recombinant protein achieved by MTT assay. In our experiment, soluble recombined RC28 proteins were prepared both in E. coli and Pichica yeast and the yields of purified protein from these two expression systems were about 3.5mg/L and 0.2mg/L, respectively. However, the recombined RC28 protein expressed from E. coli system showed poor antiviral activity while the recombined RC28 protein from Pichica yeast system showed much higher antiviral activity which was close to that of the natural protein.



Key wordsProtein expression and purification      RC28 antiviral protein      Escherichia coli expression system      Pichia yeast expression system     
Received: 22 August 2016      Published: 25 January 2017
ZTFLH:  Q819  
Cite this article:

HU Li-qiang, ZHENG Wen, ZHONG Yi, DU Dan, YANG Hao, GONG Meng. Comparison of Expression and Activity of Antiviral Protein RC28 in Escherichia coli and Pichia pastoris. China Biotechnology, 2017, 37(1): 14-20.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170103     OR     https://manu60.magtech.com.cn/biotech/Y2017/V37/I1/14

[1] 蒋军.抗病毒药物的研究进展与发展趋势.海峡药学, 2004, 16(6):1-3. Jiang J,Research progress and development trend of antiviral drugs.Strait Pharmaceutical Journal, 2004, 16(6):1-3.
[2] 刘琨,谢蓝.新型核苷类抗病毒药物的研究进展.药学学报, 2006, 41(8):689-693. Liu K,Xie L. Advances in the study of nucleoside antiviral drugs. Acta Pharmaceutica Sinica, 2006, 41(8):689-693.
[3] Gong M, Piraino F, Yan N,et al.Purification, partial characterization and molecular cloning of the novel antiviral protein RC28.Peptides, 2009, 30(4):654-659.
[4] Laemmli U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature, 1970, 227(5259):680-685.
[5] Aoki M, Tan M, Fukushima A,et al.Antiviral substances with systemic effects produced by basidiomycetes such as fomes fomentarius.Bioscience Biotechnology & Biochemistry, 1993, 57(2):278-282.
[6] Sorimachi K, Niwa A, Yamazaki S,et al.Anti-viral activity of water-solubilized lignin derivatives in vitro.Agricultural & Biological Chemistry, 1990, 54(5):1337-1339.
[7] Matsumoto Y, Sarkar G, Sommer S S,et al.A yeast antiviral protein, SKI8, shares a repeated amino acid sequence pattern with beta-subunits of G proteins and several other proteins.Yeast, 1993, 9(1):43-51.
[8] Akihisa T, Franzblau S G, Tokuda H,et al. Antitubercular activity and inhibitory effect on epstein-barr virus activation of sterols and polyisoprenepolyols from an edible mushroom, Hypsizigus marmoreus.Biological & Pharmaceutical Bulletin, 2005, 28(6):1117-1119.
[9] Mothana R A A,Ali N A A,Jansen R,et al.Antiviral lanostanoid triterpenes from the fungus Ganoderma pfeifferi.Fitoterapia, 2003, 74(1-2):177-180.
[10] Gu C Q, Li J W, Chao F,et al.Isolation, identification and function of a novel anti-HSV-1 protein from Grifola frondosa.Antiviral Research, 2007, 75(3):250-257.
[11] Wang H, Ng T B. Isolation and characterization of velutin, a novel low-molecular-weight ribosome-inactivating protein from winter mushroom (Flammulina velutipes) fruiting bodies.Life Sciences, 2001, 68(1):2151-2158.

No related articles found!