Soluble Expression And N-terminal Site-specific PEGylation Of LCVN

Cyanovirin-N is an 11-KD 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 aborts cell-to cell fusion and transmission of HIV-1 infection.Due to its cyanobacterial origin it is likely to show the typical drawbacks associated with pHarmaceutical use of foreign proteins such as short plasma half-life, proteolysis and immunogenicity. Poly (ethylene glycol)(PEG) is a widely investigated polymer used for the covalent modification of biological macromolecules for many pHarmaceutical and biotechnical applications, especially of peptides and proteins. PEGylation reagents could conjugate N-terminal residues (a-amine groups), lyscine (ε-amine) and cystine residues, but random PEGyaltion usually result in inactive molecules as multi-PEGyaltion production or PEGyaltion sites located proximal to the active site.Here, the (GGGGS)3 flexible Linker was linked to the N-terminus of CVN and constructed the vector PET3c-SUMO-LCVN. This SUMO-fused LCVN was expressed in the cytoplasm of E. coli in a folded and soluble form, up to 30% of the total soluble protein. The preparation of LCVN was very efficinent and high yield with lower cost due to SUMO-LCVN was very susceptible to SUMO protease and fusion protein could be cleaved completely without DTT, so LCVN protein was more propitious for large-scale production. Poly-ethylene glycol (PEG) with average molecular weight of 20,000 and 30,000 daltons was covalently attached to the N-terminus of LCVN. MonoPEGylated-LCVN was separated by SP sepHarose chromatograpHy. The bioactivity assay demonstrated that LCVN and monoPEGyalted-LCVN bound to gp120 with nanomolar concentration. In addition, LCVN possessed enhanced in vitro anti-HSV-land anti-HIV/ⅢB activity along with decreased cytotoxicity compared to native CVN. MonoPEGyalted-LCVN remarkerablely reduce the cell-to-cell fusion and polykaryoctyes formation, and retain significantly anti-HIV and anti-HSV-1 activity with pronounced decreased cytotoxicity.