N-acetyl-D-glucosamine -2-- epimerase Cloning Expression and Purification.docVIP

 PAGE \* MERGEFORMAT 14 N-acetyl-D-glucosamine -2-- epimerase Cloning Expression and Purification Of: Yuyun Yan, Zhang Weixing, Yan Shin, Tangqin Qin, Liu Naihua, Yanyong Bo, LI Xiao, Lin Shaoqiang [Abstract] Objective: to extract from the kidney RNA, cloned from N-Acetyl-D-glucosamine -2-- epimerase (GNE) gene in E. coli E.coli BL21 (DE3) expression. Methods: The total extracted from the kidney RNA, cloned by RT-PCR GNE gene by sequencing, transformation of E. coli E.coli DH5a, plasmid extraction, identification, and E. coli BL21 (DE3) transformed expression, through the His- tag affinity purification (NiSepharose 6 Fast Flow), G-25 desalting column, SDSelectrophoresis. Results: RNA extraction by UV spectrophotometer OD260/OD280 = 1.77, shows no significant contamination of protein and phenol. RT -PCR amplified fragment sequencing proved that the gene ORF was 1 206 bp, encoding 402 amino acids of the enzyme protein (GNE). SDSshows the purified protein was 45 kD single band, and guess the value of gene sequence . Conclusion: GNE gene was successfully cloned and expressed. [Keywords:] N-acetyl-D-glucosamine grape -2-- epimerase; gene; cloning; purification; pig Abstract: Objective: To clone the gene of N-acetyl-D-glucosamine-2-epimerase according to RNAextracted from pig kidney and express it in E.coli BL21 (DE3) and then purify the product. Methods: RNA was extracted from porcine kidney to clone the gene of N-acetyl-D-glucosamine-2-epimerase withRT-PCR followed by sequencing. Recombinant plasmid was converted into E.coli DH5a and to identifyit followed by the conversion of the recombinant plasmid in E.coli BL21 ( DE3) and expression. Theprotein was purified by His-tag (Ni Sepharose 6 Fast Flow), desalted by G-25 column and identifiedby SDS. Result: RNA was extracted from porcine kidney and identified with ultravioletspectrophotometer: OD260 = 0.0346, OD280 = 0.0195, OD260/OD280 = 1.77 (between1.7 and 2.2), it showedthat there was no protein and phen