Screening of strong promoters from corynephage P1201 for the construction of expression vectors used in the Corynebacterium glutamicum

• Main Authors: Kun-Reui Chiu, 邱堃睿
• Other Authors: 許文輝
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/17548317672917462760

碩士 === 國立中興大學 === 分子生物學研究所 === 94 === Corynebacterium glutamicum is generally recognized as nonhazardous organism, which is safe to handle. Based on its extremely well investigated central metabolism and well-established molecular biology tools, C. glutamicum may be very suitable as an organism for the expression of heterologous gene in high (G+C) bacteria. Promoter regions from putative ORFs of corynebacteriophage P1201 were cloned into 5′ end of reporter cat gene and then transformed into C. glutamicum. Promoters from the genes corresponding to ORF 24 and ORF 29 showed CAT specific activities of cat enzyme was 7.17 and 8.17 U/mg protein, repectively. DNA fragments of Sau3AI digested corynephage P1201 genomic DNA were also cloned into pORC, a promoter probed vector, with cat as a reporter gene. P′1,P′2,and P′3 fragments show cat enzymes with specific activities of 8.67, 6.28 and 3.50 U / mg protein repectively, were obtained which are higher than that of trc promoter from E.coli and gdh promoter from C. glutamicum glutamate dehydrogenase gene. Corynephage P1201 structural proteins including portal protein, tail protein, and head protein were found by SDS－PAGE analysis, LC/MS/MS analysis, and N－terminal amino acid sequencing. Putative head protein from ORF 39 exhibited different mobility in SDS－PAGE analysis indicating that proteolytic cleavge might be involved in the maturation of phage capsid protein.