[Expression, purification and activity determination of cyanovirin-N].
Cyanovirin-N (CVN) is an 11 kDa anti-HIV protein originally isolated from extracts of a cyanobacterium, Nostoc ellipsosporum. The protein binds with high affinity to the viral envelope glycoprotein gp120 and irreversibly inactivates diverse HIV strains. A fusion gene consisting of cvn, sumo and 6xHis tag was synthesized by PCR according to the codon bias of Escherichia coli. The fusion protein is expressed in the cytoplasm of E. coli in a soluble form and up to 28% of the total protein. The recombinant CVN was purified to homogeneity by 2 rounds of Ni-NTA affinity chromatography and one round of SUMO protease cleavage. Bioactivity assay demonstrated that SUMO-CVN and CVN bound to gp120 with nanomolar concentration. In addition, CVN showed potent anti-HSV-1 and anti-HIV-1 activities in in vitro cellular assays. Therefore, the 6xHis SUMO fusion expression and purification system provides a better approach for large scale production of CVN for further microbicide development.
Chen
Wei
W
Biomedical Research and Development Center, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou 510630, China.
Han
Bo
B
Qian
Chuiwen
C
Liu
Qiuying
Q
Xiong
Sheng
S
D000998:Antiviral Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects*; D018259:Herpesvirus 1, Human / Q000187:drug effects*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*
D000998:Antiviral Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; C107733:cyanovirin N