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), 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 (beta/alpha)(8)-barrel domain and conserved regions of the alpha-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 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.
Ko
Yuan-Tih
YT
Department of Food Science, Biotechnology Division, School of Life Sciences, National Taiwan Ocean University, Keelung, Taiwan.
Chung
Pei-Shan
PS
Shih
Yun-Chi
YC
Chang
Ja-Wei
JW
D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D007887:Fabaceae / Q000201:enzymology*; D015870:Gene Expression; D012091:Repetitive Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000201:enzymology; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D018076:DNA, Complementary; D015061:1,4-alpha-Glucan Branching Enzyme