α 1 2- Cloning and Expression of alpha 1 2-fucosyltransferase in E. coli BIOTECHNOLOGY BULLETIN

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1 BIOTECHNOLOGY BULLETIN α PCR Helicobacter pylori HP DNA α 1 2- α 1 2-fucosyltransferase α 1 2-fuct 906 bp pet-22b + pet-fuct BL21 DE mmol /L IPTG 4 h SDS-PAGE 33 kd α 1 2- BL21 HPLC pmol / mgpr h α 1 2- Cloning and Expression of alpha 1 2-fucosyltransferase in E. coli Jia Honghong Li Yu Liu Yihan Wang Yao Liang Xiaomei Lu Fuping Key Laboratory of Industrial Microbiology Ministry of Education College of Biotechnology Tianjin University of Science & Technology Tianjin Abstract A prokaryotic expression vector pet-fuct containing the gene of alpha 1 2-fucosyltransferase was constructed and expressed in E. coli BL21 DE3. The fuct gene was amplified from the extracted total DNA of Helicobacter pylori by PCR and was inserted into the prokaryotic expression vector pet-22b + to construct a recombinant expression vector pet-fuct. The expression of fuct gene was induced by 0. 1 mmol /L IPTG at 25 for 4 hours and SDS-PAGE analysis showed that protein with molecular weight of 33 kd was expressed in E. coli BL21. This protein had a specific activity of pmol / mgpr h which was detected by HPLC. Key words alpha 1 2-fucosyltransferase Gene cloning Prokaryotic expression E. coli jiahonghong850@ 163. com liyu@ tust. edu. cn

2 186 Biotechnology Bulletin DNA 1 μl P1 10 μmol /L 1 μl P2 10 μmol /L 1 μl dntps 2. 5 mmol /L 2 μl 10 4 buffer 2 μl Taq 0. 5 μl dh 2 O μl PCR 20 μl Helicobacter pylori HP DNA α s min pet pet-fuct PCR pet-22b + BamH I Helicobacter pylor HP BL21 DE3 DH5α pet-22b + pet-fuct SDS-PAGE Tryptone pet-fuct E. coli BL21 Yeast Extract OXOID pet-22b + IPTG TEMED Sigma Taq DNA dntps T 4 DNA ml LB 100 μg /ml Amp 37 BamH I Hind III 1% 30 ml DNA DNA 250 ml OD E. coli BL IPTG Invitrogen 0. 1 mmol /L 20 4 h DNA DNA DNA 6 SDS-PAGE Fluor Chem PCR GenBank Helico- bacter pylori α 1 2- fuct E. coli BL21 IPTG P1 5'-CATGCCATGGATCCGATGGC- TTTTAAAAGTGTGCAA-3' BamH I 95 5 min s s Hind III T 4 DNA 16 DH5α PCR SDS-PAGE 10% 5% 100 μg /ml LB r /min 1% 100 μg /ml LB OD 600 = mmol /L IPTG r /min 4 h P2 5'-CCCAAGCTTGAGCTCTTAAG- CGTTATACTTTTGGGATTT-3' Hind III DNA SDS- P1 P2 fuct PAGE IPTG

3 α IPTG h SDS-PAGE IPTG IPTG h SDS-PAGE IPTG ph W 4 s 5 s 10 min r /min 4 10 min SDS- PAGE GDP- GDP- petfuct /BL μmol /L 20 mmol /L MnCl 2 1% Triton X mmol /L - ph nmol /L -β-d mg 100 μl 37 1 h 300 μl / 2 1 V /V r /min 2 min HPLC HPLC fuct M. 1 kb DNA ladder 1. PCR fuct DNA DNA 2 pet-fuct bp 906 bp pet-22b-fuct petfuct /DH5α fuct pet-22b + 2 pet-fuct BamH I 906 bp GenBank Hind III bp bp pet-22b pet-22b + pet-fuct /BL21 pet- 22b/BL21 IPTG 0. 1 mmol /L r/min 1

4 188 Biotechnology Bulletin IPTG 0. 1 mmol /L IPTG 5 IPTG M. 1 kb DNA ladder 1. PCR fuct 2. pet-22b + /BamH I /Hind III 3. pet-fuct /BamH I /Hind III 4. pet-fuct /BamH I 3 pet-fuct 4 h SDS-PAGE 4 33 kd IPTG 0. 1 mmol /L h 25 6 fuct Marker 2. pet-22b /BL pet-fuct /BL21 4 SDS-PAGE IPTG 5 IPTG mmol /L 0. 1 mmol /L 0. 1 mmol /L 4 h IPTG 2. 6 IPTG IPTG 0. 1 mmol /L h

5 α HPLC pet-fuct / BL pmol / mg- GDP- Pr h Phenyl-β-D-Gal- α1 2- GDP pmol / mgpr h HPLC h 8 GDP GDP /4- α α1 2- GDP- 10 fuct pet-22b M. Marker pet-fuct BL21 8 DE r /min IPTG 0. 1 mmol /L SDS Fuct PAGE 1 Koizumi S Endo T Tabata K et al. Large-scale production of GDP-fucose and lewis X by bacterial coupling. Journal of Inductril Microbiogy & Biotechnology Ofek I Hasty DL Sharon N et al. Anti-adhesion therapy of bacte-

6 190 Biotechnology Bulletin rial diseases prospects and problems. FEMS Immunology and Medical Microbiology K88 9 Lin B Saito M Sakakibara Y et al. Characterization of three members of murine α 1 2-fucosyltransferases change in the expression 4 Priem B Gilbert M Wakarchuk WW et al. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology Novagen. pet System Manual 9th Edition M Sambrook J Russell D. Molecular cloning a laboratory manual M. New York Cold Spring Harbor Labouatory M Doig SD O Sullivanb LM Patel S et al. Large scale production of cyclohexanone monooxygenase from Escherichia coli TOP10 pqr239. Enzyme Microb Technol of the Se gene in the intestine of mice after administration of microbes. Archives of Biochemistry and Biophysics Tabata K Koizumi S Endo T et al. Production of UDP-N-acetylglucosamine by coupling metabolically engineered bacteria. Biotechnology Letters

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