Soluble cytoplasmic expression, rapid purification, and characterization of cyanovirin-N as a His-SUMO fusion.
Cyanovirin-N (CVN) is a promising antiviral candidate that has an extremely low sequence homology with any other known proteins. The efficient and soluble expression of biologically functional recombinant CVN (rCVN) is still an obstacle due to insufficient yield, aggregation, and abnormal modification. Here, we describe an improved approach to preparing native rCVN from Escherichia coli more efficiently. A fusion gene consisting of cvn and sumo (small ubiquitin-related modifier) and a hexahistidine tag was constructed according to the codon bias of the host cell. This small ubiquitin-related modifier (SUMO)-fused CVN is expressed in the cytoplasm of E. coli in a folded and soluble form (>30% of the total soluble protein), yielding 3 to 4 mg of native rCVN from 1 g of wet cells to a purity up to 97.6%. Matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry and reverse-phase high-performance liquid chromatographic analysis showed that the purified rCVN was an intact and homogeneous protein with a molecular weight of 11,016.68 Da. Potent antiviral activity of rCVN against herpes simplex virus type 1 and human immunodeficiency virus type 1/IIIB was confirmed in a dose-dependent manner at nanomolar concentrations. Thus, the His-SUMO double-fused CVN provides an efficient approach for the soluble expression of rCVN in the cytoplasm of E. coli, allowing an alternative system to develop bioprocess for the large-scale production of this antiviral candidate.
Gao
Xianglei
X
Biomedical R&D Center, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, Guangdong, People's Republic of China.
Chen
Wei
W
Guo
Chaowan
C
Qian
Chuiwen
C
Liu
Ge
G
Ge
Feng
F
Huang
Yadong
Y
Kitazato
Kaio
K
Wang
Yifei
Y
Xiong
Sheng
S
D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects; D018259:Herpesvirus 1, Human / Q000187:drug effects; D006639:Histidine / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019380:Anti-HIV Agents; D000998:Antiviral Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C107733:cyanovirin N; D006639:Histidine