| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 90 === N-acetyl D-glucosamine 2-epimerase (GlcNAc 2-epimerase) has been used for the synthesis of sialic acid. GlcNAc 2-epimerase gene was cloned into pQE-30 and expressed in E. coli in which only 20% of GlcNAc 2-epimerase produced was soluble form. The expression of GlcNAc 2-epimerase gene in E. coli strains JM109, XLI-Blue, and Nova Blue, was higher than that in strain DH5a. IPTG concentration and temperature (28℃and 17℃) had not significantly effect on the expression of GlcNAc 2-epimerase gene. To increase the amount of soluble GlcNAc 2-epimerase, the gene was fused to the thioredoxin gene and expressed in E. coli BL21 (DE3). However, the solubility of GlcNAc 2-epimerase was still not improved. The specific activity of the fused GlcNAc 2-epimerase decreased 10-fold in the crude cell extract. Soluble GlcNAc 2-epimerase was also not increased in the co-expression of GroES or GroEL with GlcNAc 2-epimerase or thioredoxin-GlcNAc 2-epimerase gene. When the sialic acid aldolase gene was fused to N-terminal of GlcNac 2-epimerase gene and expressed in E. col Nova Blue, no GlcNAc 2-epimerase activity was detected in the crude cell extract and all fusion proteins were insoluble. A T-vector was constructed for the rapid and selective screening of the gene which expresses soluble form protein. This vector contained 6x His tag, multiple cloning sites, and LacZ-a fragment under the control of T5 promoter of the pQE-30. Two XcmI sites in multiple cloning sites will facilitate the direct cloning of mutated genes from error-prone PCR.
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