The beginnings of mucin biosynthesis: The crystal structure of UDP-GalNAc:polypeptide α- N -acetylgalactosaminyltransferase-T1

Fritz et al. 10.1073/pnas.0405657101.

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Expression vector construction. All oligonucleotides were from Integrated DNA Technologies (Coralville, IA). Phosphorylated oligonucleotides dAATTCGACGCGT and dAATTACGCGTCG were annealed and ligated into the EcoR1 site of the vector pPIC9 (Invitrogen) to create the vector pKN55 containing a unique Mlu1 restriction site. The phosphorylated oligonucleotide dGTAAAAATCGAAGAAGGTAAACTG and a nonphosphorylated oligonucleotide dCACCACGAATTCCGCGTCTTTCAGGGC were used to PCR amplify bases 1606-2697 of the plasmid pMAL-p2 (New England Biolabs) corresponding to residues 27-390 of the Escherichia coli periplasmic maltose-binding protein (malE). The PCR product was digested with EcoR1 and cloned into the SnaB1-EcoR1 restriction sites of pKN55 to create the vector pKN55-malE. The phosphorylated oligonucleotides dAATTCTCTTCTGAAAACTTGTACTTTCAATCTGA and dCGCGTCAGATTGAAAGTACAAGTTTTCAGAAGAG containing the tobacco etch virus (TEV) protease recognition sequence ENLYFQS were annealed and cloned into the EcoR1-Mlu1 sites of pKN55-malE to create the plasmid pKN55-malE-TEV. The sequence encoding a truncated version of murine ppGaNTase-T1 between the Mlu1 and Age1 sites of the vector pIMKF3 (1) was cloned into the Mlu1-Age1 sites of pKN55-malE-TEV to create the vector pKN55-mT1-malE-TEV.

Expression screening. Electroporation-competent Pichia pastoris strain SMD1168 (Invitrogen) was prepared according to the manufacturer’s instructions. Vectors were linearized with SacI and electroporated into competent cells by using a Bio-Rad Gene Pulsar set at 1,500 V, 25 m F, and 200 ohms. Cells were grown for 3 days at 30°C on minimal dextrose plates (1.34% yeast nitrogen base, 2% dextrose, 4 × 10-5% biotin) lacking histidine. Individual colonies were grown in 2 ml of YPG-case medium (1% yeast extract/2% peptone/1.34% yeast nitrogen base/1% glycerol/1% casamino acids) in 24-well plates. Cells were grown at 250 rpm in an orbital shaker at 30°C for 18-24 h and centrifuged at 2,000 × g for 5-10 min. The supernatant was replaced with 0.4 ml of YPM-case medium (1% yeast extract/2% peptone/1.34% yeast nitrogen base/1% methanol/1% casamino acids) to induce protein expression. Cells were cultured for an additional 20-24 h at 20°C, centrifuged, and the supernatants were analyzed for protein expression by SDS/PAGE on 4–12% Bis-Tris gradient gels by using MARK-12 protein standards (Invitrogen).

Fermentation. Fermentation was performed in a 7-liter bench-top fermentor (New Brunswick Scientific) with a 5-liter working volume. The fermentor was interfaced to a MD-Biostat System (B.Braun Biotech USA, Allentown, PA), furnished with an adaptive control algorithm (2) to maintain dissolved oxygen level at 30% saturation by adjusting both agitation and the air or oxygen supply. Protein production was performed by using a fed-batch strategy based on On-Line methanol sensor (3). The fermentor with 3 liters of modified BMGY media [20 g/liter of Bacto peptone (BD Biosciences, Sparks, MD), 10 g/liter of Bacto yeast extract (BD Biosciences), 10 g/liter of Bacto casamino acids (BD Biosciences), 40 g/liter of glycerol, 13.4 g/liter of yeast nitrogen base (BD Biosciences), and 0.0001 g/liter of biotin) was inoculated with 100 ml of overnight culture and grown in batch mode for » 16 h at 30°C, pH 6 (OD » 80 at 600 nm), after which fed-batch mode was initiated by adding a 50% glycerol solution at a flow rate of 18 ml/liter per hour for another hour (OD » 100 at 600 nm). The growth temperature was than reduced to 20°C, the pH was increased to 7.0, and the glycerol feed rate was reduced gradually to 2 ml/liter per hour. Addition of methanol containing 12 ml/liter of PTMI solution (3) was commenced, and methanol concentration was controlled at 3 g/liter by using an on-line monitoring and control device described previously (4). Supernatant containing mppGaNTase-T1 was collected after 24-h methanol induction.

Purification. Chromatography columns were from Amersham Pharmacia Biosciences. Fermentor supernatants were cooled to 4°C, brought to 10 mM 2-mercaptoethanol (b -ME) and 5 mM EDTA and concentrated to 250 ml by using a Pellicon-2 Mini tangential flow concentrator (Millipore) with a 30-kDa Mr pore size. The concentrate was diafiltered against 25 mM Tris (pH 8)/10 mM b -ME (buffer A) by 12 cycles of 2-fold dilution and 2-fold concentration and applied to a Q-Sepharose HP column. The column was eluted with a five-column volume linear gradient of 0-1.0 M NaCl in buffer A. Fractions containing product were dialyzed against 25 mM Mes (pH 6.0)/10 mM b -ME/1 mM EDTA (buffer D), and applied to a SP-Sepharose HP column. The column was eluted by using a 0-1.0 M NaCl gradient in buffer D. Fractions containing product were pooled and dialyzed against 25 mM Mes (pH 6.4)/10 mM b -ME/1 mM EDTA (buffer E). During this dialysis, copurifying protease activity quantitatively cleaved the maltose-binding protein from ppGaNTase-T1 between L50 and V51 of mppGaNTase-T1 releasing fully active transferase. The dialyzed sample was reapplied to the SP-Sepharose HP column and eluted with a 0-1.0 M NaCl gradient in buffer E. Fractions containing ppGaNTase-T1 were pooled and dialyzed against 25 mM Tris (pH 7.5)/10 mM b -ME/1 mM EDTA. Samples were brought to 0.75 M (NH4)2SO4, applied to a Phenyl Sepharose fast-flow low substitution column, and eluted with a linear 0.75-0 M (NH4)2SO4 gradient. Fractions containing product were pooled and dialyzed against buffer C. The dialyzed sample was concentrated, brought to 10% (vol/vol) glycerol, quick frozen on dry ice/ethanol, and stored at -80°C.

1. Hagen, F. K. & Nehrke, K. (1998) J. Biol. Chem. 273, 8268-8277.

2. Hsiao, J., Ahluwalia, M., Kaufman, J. B., Clem, T. R. & Shiloach, J. (1992) Ann. NY Acad. Sci. 665, 320-333.

3. Trinh, L. B., Phue, J. N. & Shiloach, J. (2003) Biotechnol. Bioeng. 82, 438-444.

4. Wagner, L. W., Matheson, N. H., Heisey, R. F. & Schineder, K. (1997) Biotechnol. Technol. 11, 791-795.

This Article

  1. Published online before print October 14, 2004, doi: 10.1073/pnas.0405657101 PNAS October 26, 2004 vol. 101 no. 43 15307-15312
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