Expression, Purification And Characterization Of Polygonatum Cyrtonema Hua Lectin Ⅱ In Escherichia Coli

To express the gene coding mature peptide of Polygonatum cyrtonema Hua. lectin II, pET-P expression plasmids were constructed by inserting the cDNA fragment of the mature peptide into vector pET32a (+).Then the vectors with pET expression plasmids were transformed to E. coli BL21 (DE3). The positive clones were identified by Colony Western Blot. After E. coli was induced by IPTG, SDS-PAGE analysis showed that 29kD fusion proteins were expressed. Then the expression conditions were optimized to prevent the formation of inclusion bodies. In the upper lysed cells, recombinant proteins with His₆-tag in their N-terminals were purified by metal affinity chromatography first. After that, the tags can be cleaved from the fusion proteins by enterokinase. Finally, the purified recombinant PCLII were obtained (abbrev. rPCLII) by metal affinity chromatography again and followed by ion exchange chromatography. Western Blot showed that the purified rPCLII can react with RGS-His and rabbit antiserum of purified natural PCLII (abbrev. nPCLII). It suggested that rPCLII had high identity as...