Construction of a mungbean (Vigna radiata L.) starch phosphorylase cDNA in E. coli system

• Main Authors: Wei-Chi Chang, 張瑋齊
• Other Authors: Yuan-Tih Ko
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/20361438425510788682

碩士 === 國立臺灣海洋大學 === 食品科學系 === 97 === Starch phosphorylase ( SP, EC 2.4.1.1 ) is commonly found in plants and is one of the vital enzymes in the starch metabolic pathway. It catalyzes a reversible reaction between synthesis and degradation of starch with bidirectional activities. Previous studies have identified the fragments of mungbean 105-kDa SP by MALDI-TOF, conducted immunological analysis and amplified the full-length cDNA fragment of mungbean (Vigna radiata L.) SP (named VrSP). The objective of this study was to clone the sequence into an expression vector and further express into a biological-active recombinant protein in E. coli system. Its 3-D structure and functional features were predicted in silico by SWISS-Model. One matched template, rabbit muscle phosphorylase with potent inhibitor (IWW2A), predicted the N-terminal of VrSP from 92 residue to 411 residue, including partial starch-binding site. The other template, rabbit muscle phosphorylase (2GJ4A), predicted the C-terminal of VrSP from 585 residue to 984 residue, including catalytic site (711-727). VrSP insert was prepared by PCR using primers designed with EcoRI and SalI sites in the flanking region, and was ligated into the parallel sites on pMal-C2X vector, followed by transforming into Novablue cloning host and screening. Insert size of one recombinant plasmid was nearly closed to the expected 2.9 Kb, however after sequencing, 97% of its sequence is matching to the DNA sequences in E. coli cloning host. In addition, the other two major groups of recombinant plasmid had 2.2 kb and 1.4 kb insert fragments in pMal-C2X vector. It showed that the sequence of VrSP itself may cause recombination with the host E. coli chromosomal DNA during the growth of these recombinant clones.