牛肉鮮味??基因之選殖與表現

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题名:	牛肉鮮味??基因之選殖與表現
其它题名:	Cloning and Expressing of Beefy Meaty Peptide Gene
作者:	林錚威 Lin, Cheng-Wei
贡献者:	李根永 Li, Ken-Yuon 東海大學食品科學系
关键词:	牛肉鮮味??;重組蛋白 beefy meaty peptide;recombinant;umami
日期:	2002
上传时间:	2011-05-19T05:47:05Z (UTC)
摘要:	本研究是利用DNA重組技術構築一原核表現系統來生產具有類似味精風味的牛肉鮮味短鏈??。實驗中依據牛肉鮮味??的八個胺基酸殘基序列，設計兩段互補的牛肉鮮味??核?酸序列（BMP1 & BMP2），利用基因重組的技術將此核?酸序列和表現載體pEZZ18接合在一起，形成能生產牛肉鮮味??的基因載體（pEZZ18-BMP），再將此基因載體轉型入宿主細胞E.coli JM105中，以培育出能合成牛肉鮮味??的菌株。篩選出的菌株經液體培養後，利用親和性層析管柱回收具有牛肉鮮味??的融合蛋白，進一步將此融合蛋白以CNBr切割後，利用HPLC分離、純化融合蛋白中的牛肉鮮味??，並輔以胺基酸分析儀及胺基酸定序儀鑑定分離出的牛肉鮮味??其正確性。結果顯示，本實驗所構築之原核表現系統所生產之牛肉鮮味??，其胺基酸組成份與胺基酸序列皆與原始的牛肉鮮味??相同。 In this study, DNA recombinant technique was performed to construct a prokaryotic expression vector. The recombinant vector confers the transformed host cell the ability to produce beef meaty peptide. In this experiment, two complementary polynucleotides encoding beef meaty peptide were synthesized. The double strand polynucleotides with Sal 1 sticky end were inserted in pEZZ18, and then the recombinant plasmid（pEZZ18-BMP）were transformed to E.coli JM105. Some transformed E.coli colonies with the ability to produced umami peptide were isolated. The transformed E.coli were cultivated in LB broth, and the culture contained some umami peptide. The umami peptide linked with ZZ protein was isolated with an affinity chromatography. The isolated fusion protein was treated with CNBr to release the umami peptide. The umami peptide was purified with an HPLC system. The amino acid composition of the peptide was determined as those extracted from beef. The amino acid sequence was identified as that originally determined by Yamasaki and Maekawa.
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