Enhancement of Cytoplasmic Solubility of Heparinase I from Flavobacterium Heparinum in Escherichia Coli by Adding a Polycationic Peptide Tag and Co-Expression with CpkA

Objective: Heparinase is essential for heparin production and application. HepA derivedfrom Flavobacterium heparinum, is the most widely studied heparinase I. Recombinant HepA is difficult to express as its soluble form in Escherichia coli. For this reason, many studies have attempted to grow the addition of different kinds of labels at the N-terminus can help increase the solubility of HepA in the cytoplasm. In this study, we engineered a polycationic six-peptide tag, called an "anchor tag" (GGRRGR),with increased affinity for the chaperonin CpkA from Thermococcus kodakarensis, in an attempt to increase the cytoplasmic solubility of HepA. Firstly, HepA was fused with GGRRGR at its N-terminus and then subcloned in the plasmid pET21a, resulting in pET21a-HepA'. The modified HepA (HepA') was then expressed alongside pACYCDuet-1-CpkA. The results revealed that the amount of insoluble HepA' was reduced while the amount of soluble HepA' increased to 95% of the total soluble fraction of E. coli lysate. Furthermore, the specific activity of HepA' in the crude enzyme extract was 48.5 IU/mg, suggesting that the crude HepA' was almost ready for catalyzing. The specific activity of HepA' after purification (~146 IU/mg) was comparable to that of native HepA (~130 IU/mg), indicating that the polycationic peptide tag does not interfere with heparinase activity. In summary, our results showed that the co-expression of polycationic peptide tag-fused HepA with CpkA resulted in an active and soluble form of heparinase.