Cloning, characterization, and expression of mungbean ( Vigna radiata L.) starch branching enzyme II cDNA in Escherichia coli.

Full-length starch branching enzyme II (SBE, EC 2.4.1.18) cDNA from mungbean (Vigna radiata L. cv. Tainan no. 5), Vrsbell, was cloned, characterized, and expressed as an active enzyme in Escherichia coll. 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). Vrsbell 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 Vrsbell into SBE family A. Its partial 3D structure and functional features were predicted. Vrsbell has a shorter N-terminal among SBEs; however, two 6 bp (CCAGTT) direct repeat sequences (DRS) were found. A 24 bp shortened Vrsbell at the 3' end, skipping one DRS, was ligated into pET21b vector and expressed as His 6 -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.