BIOTECHNOLOGY BULLETIN 2011 3 α 1 2-300457 PCR Helicobacter pylori HP DNA α 1 2- α 1 2-fucosyltransferase α 1 2-fuct 906 bp pet-22b + pet-fuct BL21 DE3 25 0. 1 mmol /L IPTG 4 h SDS-PAGE 33 kd α 1 2- BL21 HPLC 13. 21 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 300457 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 13. 21 pmol / mgpr h which was detected by HPLC. Key words alpha 1 2-fucosyltransferase Gene cloning Prokaryotic expression E. coli 1 2 3 2010-08-11 20806060 20080601 20080435 E-mail jiahonghong850@ 163. com E-mail liyu@ tust. edu. cn
186 Biotechnology Bulletin 2011 3 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 12. 5 μl PCR 20 μl Helicobacter pylori HP DNA α 1 2-72 60 s 30 72 10 min pet 5 1. 2. 3 pet-fuct PCR pet-22b + BamH I 1 1. 1 1. 1. 1 Helicobacter pylor HP BL21 DE3 DH5α pet-22b + pet-fuct 1. 2. 4 SDS-PAGE 7 8 1. 1. 2 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 600 0. 6 - E. coli BL21 5 1. 0 IPTG Invitrogen 0. 1 mmol /L 20 4 h 1. 2 1. 2. 1 DNA DNA 1. 2. 5 DNA 6 SDS-PAGE Fluor Chem 5500 1. 2. 2 PCR GenBank Helico- bacter pylori α 1 2- fuct 1. 2. 6 E. coli BL21 IPTG P1 5'-CATGCCATGGATCCGATGGC- TTTTAAAAGTGTGCAA-3' BamH I 95 5 min 95 45 s 55 50 s Hind III T 4 DNA 16 DH5α PCR SDS-PAGE 10% 5% 100 μg /ml LB 37 180 r /min 1% 100 μg /ml LB OD 600 = 0. 6-1. 0 0. 05 0. 1 0. 25 0. 5 1. 0 1. 5 mmol /L IPTG 20 120 r /min 4 h P2 5'-CCCAAGCTTGAGCTCTTAAG- CGTTATACTTTTGGGATTT-3' Hind III DNA SDS- P1 P2 fuct PAGE IPTG
2011 3 α1 2-187 IPTG 20 25 28 30 37 4 h SDS-PAGE IPTG IPTG 2 3 4 5 6 7 8 h SDS-PAGE IPTG 1. 2. 7 1. 2. 4 ph 7. 2 400 W 4 s 5 s 10 min 12 000 r /min 4 10 min SDS- PAGE 1. 2. 8 9 GDP- GDP- petfuct /BL21 0. 37 μmol /L 20 mmol /L MnCl 2 1% Triton X-100 50 mmol /L - ph5. 8 0. 38 nmol /L -β-d- 0. 4 mg 100 μl 37 1 h 300 μl / 2 1 V /V 2 000 r /min 2 min HPLC HPLC 2 2. 1 fuct M. 1 kb DNA ladder 1. PCR fuct DNA DNA 2 pet-fuct 1 750-1 000 bp 906 bp 2. 3 2. 2 pet-22b-fuct petfuct /DH5α fuct pet-22b + 2 pet-fuct BamH I 906 bp GenBank Hind III 1 000 bp 5 400 bp pet-22b + 2. 4 3 pet-22b + pet-fuct /BL21 pet- 22b/BL21 IPTG 0. 1 mmol /L 20 120 r/min 1
188 Biotechnology Bulletin 2011 3 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 3-6 2 2. 5. 2 37 IPTG 0. 1 mmol /L 20 25 28 30 37 4 h 25 6 fuct 25 1. Marker 2. pet-22b /BL21 3-6. pet-fuct /BL21 4 SDS-PAGE 2. 5 2. 5. 1 IPTG 5 IPTG 0. 05 mmol /L 0. 1 mmol /L 0. 1 mmol /L 4 h IPTG 2. 6 IPTG IPTG 0. 1 mmol /L 6 2. 5. 3 7 4 h
2011 3 α1 2-189 HPLC pet-fuct / BL21 13. 21 pmol / mg- GDP- Pr h Phenyl-β-D-Gal- α1 2- GDP- 13. 21 pmol / mgpr h HPLC 3 7 25 4 h 8 GDP- 4 6- GDP-4- -6-3 5- /4- α1 2-6 6 α1 2- GDP- 10 fuct pet-22b M. Marker 1. 2. + pet-fuct BL21 8 DE3 25 120 r /min IPTG 0. 1 mmol /L SDS- 2. 7 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 2000 25 4 213-217. 2 Ofek I Hasty DL Sharon N et al. Anti-adhesion therapy of bacte-
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