| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 96 === Glycinins, accounting for 40% of the total seed proteins, are the predominant storage proteins in soybean seeds. It is a hexameric protein composed of five major subunits and encoded by seven genes (gy1~gy7). The aims of this study were to enhance the physicochemical values of soybean seed proteins by genetic modification of gy5 with multiple copies of bioactive peptide sequences (VVYP). Three sites of the variable region (I, II, and III) in the Gy5 protein available for genetic manipulation were predicted by computer simulation to reduce the possible change of the three-dimensional structures of the modified proteins and used in this study. A total of twelve different gy5 mutants with one to five tandem repeat of VVYP codons in the variable regions were generated by crossover PCR or annealing of the designed primers. Results revealed that modification in site III showed the best performance in expression level and solubility of the modified Gy5 proteins in E. coli, followed by modification in site II. Three mutants containing eight, nine and ten copies of VVYP sequences were also constructed. They were generated from a combination of five copies of VVYP sequences at site II and three, four, and five copies of VVYP sequences at site III, respectively. The mutant with eight copies of VVYP, Gy5-v2533, showed the best balance of the expression level and solubility under the conditions we tested. A total of fifteen modified Gy5 proteins were over-expressed in E. coli and then purified from the inclusion bodies. Yields of VVYP peptide released by in vitro digestion of these modified Gy5 proteins with trypsin and carboxypeptidase B were analyzed by HPLC. Results revealed that the VVYP peptide released from the modified Gy5 proteins is proportion to the copy number of VVYP sequences introduced into the variable region. Overall, the yield of mutant Gy5-v2534 was the best among those mutants and approximately 7159.93 pmol of released VVYP peptide could be obtained from 50 ug of purified protein.
It has been of continuing interest in identifying and developing protein source as alternatives to fish meal for use within aquafeeds. The second part of this study using the same approach to manipulate and modify the gy5 gene and our aim is to improve the nutritional qualities by incorporating more methionine and glycine codons. A tandem repeat DNA corresponding to the MGKMGR peptide sequences were generated by Template-repeat PCR. Sequence analyses revealed that DNA fragments containing one, six, ten, and thirteen tandem repeat of the MGKMGR peptide sequences, respectively, were obtained. These DNA fragments with the exception of thirteen copies cassette were individually incorporated into the three variable regions of the gy5 gene. In addition, a DNA fragment containing ten tandem repeat RGMKGM peptide sequence provided by Dr. W. H. Hsu was separately cloned into the three sites of variable region. The modified gy5 genes were then expressed in E. coli and only five (Gy5-M16, Gy5-M21, Gy5-M26, Gy5-M36 and Gy5-G3) among a total of twelve tested strains displayed overexpressed modified proteins and could be purified from insoluble fractions, except Gy5-G3. The MGK peptide peptide released by in vitro digestion of the purified modified Gy5 proteins with trypsin was identified by HPLC.
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