Cloning,expression and activity analysis of a novel serine protease from marine <em>Coccolithovirus</em>

Li Lihua; Qiu Jianjian; Cai Yiqin; Yu Peng; Liu Jingwen

doi:10.3969/j.issn.0253-4193.2014.08.011

Article Contents

Article Navigation > Haiyang Xuebao > 2014 > 36(8): 101-110

Li Lihua, Qiu Jianjian, Cai Yiqin, Yu Peng, Liu Jingwen. Cloning,expression and activity analysis of a novel serine protease from marine Coccolithovirus[J]. Haiyang Xuebao, 2014, 36(8): 101-110. doi: 10.3969/j.issn.0253-4193.2014.08.011

Citation:

Li Lihua, Qiu Jianjian, Cai Yiqin, Yu Peng, Liu Jingwen. Cloning,expression and activity analysis of a novel serine protease from marine Coccolithovirus[J]. Haiyang Xuebao, 2014, 36(8): 101-110. doi: 10.3969/j.issn.0253-4193.2014.08.011

Citation:

PDF( 5930 KB)

Cloning,expression and activity analysis of a novel serine protease from marine Coccolithovirus

doi: 10.3969/j.issn.0253-4193.2014.08.011

College of Food and Biological Engineering, Jimei University, Xiamen 361021, China;Research center of food microbiology and enzyme engineering thchndogy of Fujian province, Xiamen 361021, Chine

Received Date: 2013-11-14
Rev Recd Date: 2013-12-19

Abstract

Abstract

A serine protease (Sp) gene (GenBank accession number: KC161207) was cloned from the genome of marine microalgal Emiliania huxleyi virus (Coccolithovirus) EhV99B1 isolate. Bioinformatic analysis was preformed on its open reading frame and the recombinant protein was expressed in E. coli and purified by affinity chromatography. Results showed that the length of the ORF of EhV99B1-Sp was 1 110 bp,which encoded a protein of 386 amino acids with a molecular mass of 39.5 kDa and pI of 6.255. EhV99B1-Sp shared a high sequence similarity with EhV86-Sp,between whom the similarities of nucleotide sequences and deduced amino acid sequences were 95% and 97%,respectively. However,the sequence similarity between EhV99B1-Sp and serine protease from other organisms was only 28% to 32%. Sequences analysis suggested that EhV99B1-Sp might be a new member of the serine protease family. Secondary structure prediction showed that EhV99B1-Sp protein contained two motifs of typical serine protease catalytic active sites LTAGHC (histidine active site) and AICNGDSGGPLF (serine active site),and two transmembrane domains. A 60 kDa recombinant protein of EhV99B1-Sp was expressed in E. coli and confirmed by myofibril-bound serine proteinase (MBSP) antibody. The recombinant protein had normal biological function in E. coli. These data laid a foundation for further studies of EhV99B1-Sp on regulation of virus-host interaction and its application.
- Emiliania huxleyi virus (EhV),
- serine protease (Sp),
- gene clone,
- recombinant expression,
- activity analysis

FullText(HTML)

References(27)

References

Rohwer F,Thurber R V. Viruses manipulate the marine environment[J]. Nature,2009,459(7244): 207-212.

Lindell D,Jaffe J D,Coleman M L,et al. Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution[J]. Nature,2007,449(7158): 83-86.

Bidle K D,Varid A. A chemical arms race at sea mediates algal host-virus interactions[J]. Curr Opin Microbiol,2011,14(4): 449-457.

Vardi A,Van Mooy B A S,Fredricks H F,et al. Viral glycosphingolipids induce lytic infection and cell death in marine phytoplankton[J]. Science,2009,326(5954): 861-865.

Castberg T,Thyrhaug R,Larsen A,et al. Isolation and characterization of a virus that infects Emiliania huxleyi (Haptophyta)[J]. J Phycol,2002,38(4): 767-774.

Simpson A A,Nandhagopal N,Van Etten J L,et al. Structural analyses of Phycodnaviridae and Iridovirdae[J]. Acta Crystallogr,Sect D: Biol Crystallogr,2003,59(12): 2053-2059.

Kegel J U,Blaxter M,Allen M J,et al. Transcriptional host-virus interaction of Emiliania huxleyi (Haptophyceae) and EhV-86 deduced from combined analysis of expressed sequence tags and microarrays[J]. Eur J Phycol,2010,45(1): 1-12.

Wilson W H,Schroeder D C,Allen M J,et al. Complete genome sequence and lytic phase transcription profile of a Coccolithovirus[J]. Science,2005,309(5737): 1090-1092.

汪世华,王文勇,黄益洲,等. 丝氨酸蛋白酶的研究进展[J]. 福建农业学报,2007,22(4): 453-456.

Suzuki C K,Rep M,Van Dijl J M,et al. ATP-dependent proteases that also chaperone protein biogenesis[J]. Trends Biochem Sci,1997,22(4): 118-123.

Kalisz H M. Microbial proteinases[J]. Adv Biochem Eng/Biotechnol,1988,36: 1-65.

Gottesman S,Maurizi M R. Regulation by proteolysis: energy-dependent proteases and their targets[J]. Microbiol Rev,1992,56(4): 592-621.

Nierman W C,Pain A,Anderson M J,et al. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus[J]. Nature,2005,438(7071): 1151-1156.

崔士超,谢利娟,吴红芝. 丝氨酸蛋白酶与植物器官衰老关联的研究进展[J]. 安微农业科学,2010,38(15): 7742-7743,7745.

张彦锋,刘静雯,张稚兰,等. 海洋球石藻(Emiliania huxleyi)病毒硫氧还蛋白(TRX)基因的克隆及生物信息学分析[J]. 海洋与湖沼,2010,41(2): 293-300.

Sambrook J, Russell D W.分子克隆实验指南[M]. 3版. 黄培堂,译. 北京:科学出版社,2002.

季星来,孙之荣. 基于结构的丝氨酸蛋白酶超家族进化分析[J]. 电子学报,2001,29(12A): 1756-1758.

黄娟,杨维青,赵祖国. 细菌生物被膜菌群间遗传物质的转移[J]. 中国抗生素杂志,2013,38(2): 98-101.

Lopez J F,Shi Y,Tyson G W,et al. Microbial community gene expression in ocean surface waters[J]. Proc Natl Acad Sci USA,2008,105(10): 3805-3810.

La Scola B,Desnues C,Pagnier I,et al. The virophage as a unique parasite of the giant mimivirus[J]. Nature,2008,455(7209): 100-104.

Gonzlez J M,Esteban M. A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history[J]. Virol J,2010,7: 59-70.

Solioz M,Yen H C,Marris B. Release and uptake of gene transfer agent by Rhodopseudomonas capsulata[J]. J Bacteriol,1975,123(2): 651-657.

Biers E J,Wang K,Pennington C,et al. Occurrence and expression of gene transfer agent genes in marine bacterioplankton[J]. Appl Environ Microbiol,2008,74(10): 2933-2939.

Monier A,Pagarete A,Vargas C,et al. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus[J]. Genome Res,2009,19(8): 1441-1449.

Bidle K D,Haramaty L,Ramos J B,et al. Viral activation and recruitment of metacaspases in the unicellular coccolithophore,Emiliania huxleyi[J]. Proc Natl Acad Sci USA,2007,104(14): 6049-6054.

Pagarete A,Lanzen A,Puntervoll P,et al. Genomic sequence and analysis of EhV-99B1,a new Coccolithovirus from the Norwegain Fjords[J]. Intervirology,2013,56(1): 60-66.

Gaines P J,Sampson C M,Rushlow K E,et al. Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis[J]. Insect Mol Biol,1999,8(1): 11-22.

Relative Articles

Supplements(0)

Cited By

Proportional views