Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichiapastoris -- Data Supplement - Supporting Text -- Proceedings of the National Academy of Sciences

Supporting information for Choi et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0931263100

Supporting Methods
Construction of Integration Vectors.
To create an integration plasmid containing a HIS4 marker, pJN261, which is a pUC19-based plasmid containing a NotI, PacI multiple cloning site flanked by the Pichia pastoris GAPDH promoter and the Saccharomyces cerevisiae CYC1 terminator, was cut with EcoICRI (Promega) and a 2.7-kb fragment of the P. pastoris HIS4 gene that had been amplified by using the oligonucleotides 5'-GCCCAAGCCGGCCTTAAGGGATCTCCTGATGACTGACTCACTGATAATAAAAATACGG-3' and 5'-GGGCGCGTATTTAAATACAGTGGATCTATCGAATCTAAATGTAAGTTAAAATCTCTAA-3', digested with NgoMIV and SwaI, and blunt-ended by using T4 DNA polymerase was ligated into the open site. This plasmid was named pJN337. To construct a plasmid with a multiple cloning site suitable for fusion-library construction, pJN337 was cut with NotI and PacI, and two oligonucleotides (5'-GGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT-3' and 5'-TAAGGCGCGCC GAATTCATTTAAATCTGCAGGGC-3') that had been annealed in vitro were ligated into the open sites to create pJN347. To create an ARG4-marked integration plasmid, pJN261 was digested with BamHI, and the DNA fragment containing the GAPDH/CYC1 expression cassette was blunt-ended by using T4 DNA polymerase. This fragment then was cloned into pBLArg-SX (1) that had been digested with NheI and SpeI and blunt-ended to yield pJN336. After digestion with NotI and PacI, the two oligonucleotides that had been annealed in vitro (5'-GGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT-3' and 5'-TAAGGCGCGCC GAATTCATTTAAATCTGCAGGGC-3') were ligated into the open sites to create pJN346. Generation of Fusion Constructs.
A region comprising the first 90 bp of the S. cerevisiae MNS1 gene was amplified by using the oligonucleotides 5'-AAACATTGGCGGCCGCCACCATGAAGAACTCTGTCGGTATTTCAATTGCAACC-3' and 5'-GGCGCGCCCTCTCTCAAAGTGTTCGTACCATGGCACATAG-3' and cloned into the plasmid pCR2.1-TOPO (Invitrogen) to generate pJN278. In the same way the first 120 bp of the S. cerevisiae MNN9 gene was cloned by using oligonucleotides 5'-GAATAAAAGCGGCCGCCACCATGTCACTTTCTCTTGTATCGTACCGCCTAAGAAAGAACCCG-3' and 5'-GGCGCGCCCCAACGAGATTGATCTCTCTGGAAAAAAATTATATATATTAG-3', and the first 219 bp of the S. cerevisiae MNN10 gene were cloned by using oligonucleotides 5'-TGCATGTAGCGGCCGCCACCATGTCTAGTGTACCTTATAATTCCCAACTTCCTATATCC-3' and 5'-GGCGCGCCCGCTTATGGTGGAATCGCTTATCCATATGAAT-3', creating plasmids pJN271 and pJN382, respectively. The ORF of a putative Caenorhabditis elegans a -1,2-mannosidase (EMBL Q90787) was amplified from a C. elegans cDNA library (gift from Robert Barstead, Oklahoma Medical Research Foundation, Oklahoma City) by using oligonucleotides 5'-ATGGGCCTCCGATCACACGAACAACTTGTC-3' and 5'-TTAATGCCGAATGCGGAATGGATGAGCTTC-3'. A single clone was isolated, sequenced, and used to make two truncation constructs by PCR with Pfu Turbo polymerase (Stratagene). The first truncation construct, which is missing the first 93 bp, was amplified by using oligonucleotides 5'-CTTGGCGCGCCGGCGGCGCTGATCTGTATTTC-3' and 5'-CTTTTAATTAATTAATGCCGAATGCGGAATGG-3', and the second truncation construct, which is missing the first 240 bp, was amplified by using oligonucleotides 5'-CTTGGCGCGCCGGAGCATCAGATGATGCAGAA-3' and 5'-CTTTTAATTAATTAATGCCGAATGCGGAATGG-3'. The amplified products were inserted into vector pJN347 by using AscI and PacI sites, creating pRCD127 and pRCD128, respectively. Plasmid pJN382 was digested with NotI and AscI, and the 219-bp insert was ligated into pRCD127 to create an in-frame fusion of the MNN10 transmembrane domain with the C. elegans mannosidase 1B (pBB27). In the same way the transmembrane domain of MNS1 from pJN278 was ligated into pRCD128 to create pBC4. Plasmid pPB103, containing the Kluyveromyces lactis MNN2-2 gene, encoding a Golgi UDP-N-acetylglucosamine transporter, was constructed by cloning a blunt BglII–HindIII fragment from vector pDL02 (kindly provided by Carlos B. Hirschberg, Boston University, Boston) into BglII- and BamHI-digested and blunt-ended pBLADE-SX (gift from James M. Cregg, Keck Graduate Institute, Claremont, CA) (1). A portion of the gene encoding human N-acetylglucosaminyltransferase I (MGATI, GenBank accession no. NM-002406), lacking the first 112 bp, was amplified by PCR with oligonucleotides 5'-TGGCAGGCGCGCCTCAGTCAGCGCTCTCG-3' and 5'-AGGTTAATTAAGTGCTAATTCCAGCTAGG-3' and vector pHG4.5 (ATCC no. 79003) as template. The resulting PCR product was cloned into pCR2.1-TOPO, and the correct sequence was confirmed. After digestion with AscI and PacI, the truncated b -1,2-N-acetylglucosaminyltransferase I (GnTI) was inserted into plasmid pJN346 to create pNA. After digestion of pJN271 with NotI and AscI, the 120-bp insert was ligated into pNA to generate an in-frame fusion of the MNN9 transmembrane domain with the GnTI, creating pNA15.

1. Lin Cereghino, G. P., Lin Cereghino, J., Sunga, A. J., Johnson, M. A., Lim, M., Gleeson, M. A. & Cregg, J. M. (2001) Gene 263, 159–169.