Cloning, Characterization, and Expression of Mungbean (Vigna radiata L.) Starch Branching Enzyme II cDNA in Escherichia coli
| Autor: | Yun-Chi Shih, Ja-Wei Chang, Pei Shan Chung, Yuan-Tih Ko |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
DNA
Complementary Starch Radiata Gene Expression Sequence Homology Biology Molecular cloning medicine.disease_cause Vigna chemistry.chemical_compound 1 4-alpha-Glucan Branching Enzyme Complementary DNA Escherichia coli medicine Amino Acid Sequence Amylase Cloning Molecular Repetitive Sequences Nucleic Acid chemistry.chemical_classification Base Sequence Reverse Transcriptase Polymerase Chain Reaction food and beverages Fabaceae Sequence Analysis DNA General Chemistry biology.organism_classification Molecular biology Enzyme chemistry Biochemistry Seeds biology.protein General Agricultural and Biological Sciences |
| Zdroj: | Journal of Agricultural and Food Chemistry. 57:871-879 |
| ISSN: | 1520-5118 0021-8561 |
| DOI: | 10.1021/jf802544b |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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