High yield production of recombinant cyanovirin-N (antiviral lectin) exhibiting significant anti-HIV activity, from a rationally selected Escherichia coli strain

Abstract

Cyanovirin-N (CV-N), a lectin of cyanobacterial origin, has important medical implications due to its highly effective virucidal activity. However, its low production yield has limited its application till date. In this study, we demonstrate for the first time, a simple process for a break-through production of soluble and bioactive recombinant CV-N (rCV-N) in a newer strain called Escherichia coli SHuffle® T7 Express lysY that is engineered for enhanced production of correctly disulphide bonded proteins. As rCV-N contains two critical disulphide bonds required for its anti-HIV activity, rational choice of this expression host could produce high yield of soluble rCV-N (∼24 mg/ 100 ml) in simple Erlenmeyer flask at 20 °C. The protein could be obtained to near purity in single-step affinity purification (mixture of monomer, dimer and higher order oligomers together referred to as mixed form) which was further resolved into monomer and dimer by size exclusion chromatography. The purified rCV-N had a monomeric mass of 11.962 kDa and a prominent β-sheet secondary structure. The three forms of rCV-N i.e. mixed, monomer and dimer exhibited significant anti-HIV activity (IC50 0.5−5 nM) and a therapeutic index of ∼1,000–10,000 in vitro (negligible cytotoxicity up to 5 μM). All rCV-N forms had low endotoxins.