Cloning, Characterization, and Expression of Mungbean (Vigna radiata L.) Starch Branching Enzyme II cDNA in Escherichia coli

Abstract

Full-length starch branching enzyme II (SBE, EC 2.4.1.18) cDNA from mungbean (Vigna radiata L. cv. Tainan no. 5), VrsbeII, was cloned, characterized, and expressed as an active enzyme in Escherichia coli. Gene-specific primers first amplified an internal cDNA by reverse transcriptase Polymerase Chain Reaction (RT-PCR), followed by obtaining 5′ and 3′ fragments by RT-PCR and rapid amplification of cDNA ends (RACE). VrsbeII possesses a complete open reading frame (ORF) of 2571 bp, and the deduced polypeptide includes the common catalytic (β/α)8-barrel domain and conserved regions of the α-amylase family. Phylogenetic analysis classified VrsbeII into SBE family A. Its partial 3D structure and functional features were predicted. VrsbeII has a shorter N-terminal among SBEs; however, two 6 bp (CCAGTT) direct repeat sequences (DRS) were found. A 24 bp shortened VrsbeII at the 3′ end, skipping one DRS, was ligated into pET21b vector and expressed as His6-rVrSBEII in E. coli BL21 (DE3) cells. The optimal expression condition for rVrSBEII was evaluated and detected by Western blot with a molecular size of 108 kDa and activity of 6.4 U/mg.