Journal of Pure and Applied MicrobiologyVol. 9 No. 1

Purification, Activity Assay and Optimization of Antarctic Algae Chlamydomonas sp. ICE-L Photolyase Expressed in E.coli

Qi Xiaoqing1#, Wang Shuai2#, Chen Hao3, Li Chongjie1, He Yingying1, Zheng Zhou1, Wang Yibin1, Liu Fangming1, Miao Jinlai1*

1 Key Lab of Marine Bioactive Substances, The First Institute of Oceanography, State Oceanic Administration (SOA), No. 6 Xianxialing Road, Qingdao - 266061, China. 2Marine College, Shandong University at Weihai, No. 180 Wenhuaxi Road, Weihai - 264209, China. 3Medical College, Qingdao University, No. 308 Ningxia Road, Qingdao - 266071, China.

Received on 20 November 2014 and accepted on 08 January 2015

 

ABSTRACT

Chlamydomonas sp. ICE-L is a type of ice algae capable of withstanding the strong ultraviolet radiation in Antarctic regions. Existing research reveals that DNA photolyase is the key compound responsible for resisting UV-B radiation, repairing DNA damage, and maintaining hereditary stability. We have sequenced and analyzed the transcriptome of Chlamydomonas sp. ICE-L and identified the cyclobutane pyrimidine dimmer(CPD) gene fragment. The full-length cDNA sequence has been determined by the RACE method and analyzed using bioinformatics software. As well, a CPD photolyase recombinant gene bacteria have been engineered. To improve the protein concentration and lay foundation for further studies of photolyase function in cells and mouse skin, this article focuses on purifying the protein, sequencing the protein N-terminal, and confirming the optimal conditions for PHR2 expression. N-terminal sequencing identified the five amino acids at the N-terminal as D/Q/T/A P K R T. Response surface methodology assays confirmed the optimal conditions for recombinant bacteria expressing photolyase to be a pH of 6.98, salinity of 1.40%, 22.01 ?C, and IPTG concentration of 0.53 mmol/L.

Keywords : Chlamydomonas sp. ICE-L, photolyase, E.coli, activity assay, expression optimization.