Heterologous Protein Secretion by Candida Utilis

Electronic Supplementary Material

Appl Microbiol Biotechnol

Heterologous protein secretion by Candida utilis

Maya Kunigo, Christoph Buerth, Denis Tielker and Joachim F. Ernst*

Department Biologie, Molekulare Mykologie, Heinrich-Heine-Universität, Düsseldorf, Germany

*Corresponding author:

Joachim F. Ernst

Department Biologie

Molekulare Mykologie

Heinrich-Heine-Universität

Universitaetsstrasse 1/26.12.01

40225 Düsseldorf,

Germany

tel/fax: +49-211-8115176

Table S1. List of oligonucleotides

Name / Sequence
CaARS-KpnI/for / 5´-ATTGGTACCTCCGGGGTAGCGATGAG-3´
CaARS-KpnI/rev / 5´-ATTGGTACCGTCCAGGACCGGCCAGAC-3´
TDH3p Rev / 5´-CCAAGCTTTATGTTGTTTGTAAGTGTG-3´
TDH3p Fwd / 5´-TTGGCGCCCTTACAGCGAGCACTCAAATC-3´
Gas1 Fwd / 5´-CCCAAGCTTAATAATGAGATTCAACTGGGCCTTGGCTTCTGCGTTCGCTG-3´
Gas1 Rev / 5‘-GATATCTCAGAACAAGAAAGCACCCATGCTAACTGTGAAGAAAGTACCG-3´
CalB his Fwd / 5´-CGCGGCCGCATATCTCATCATCATCATCATCATTGAGCAT-3´
CalB his Rev´ / 5´-GCTCAATGATGATGATGATGATGAGATATCGCGGCCGCGAT-3´
CalB Nhe1 Fwd / 5´-CTAGCTAGCGCTACCCCATTGGTCAAG-3´
CalB Nhe1 Rev / 5´-CTAGCTAGCTGCGAAGTGGCGCGCCTC-3´
SP-GPI Fwd / 5´-GCTAGCTCAACTTCTTCATCTACCG-3´
SP-GPI Rev / 5´-CTGGTTAGCAAGAGCAAC-3´
CalB PICZ Fwd / 5´-CGAATTCAATAATGGAAGTTGTTGTCCTTGAC-3´
CalB PICZ Rev / 5´-AAGGGCCCTGGGGTGACGATACCAGAACA-3´
INV-SP-FWD / 5‘-GCaagcttATGTCGTTGACAAAAG-3‘
INV-SP-REV / 5‘-CGgatatcGGAATCAACTAAACTAG-3‘
PrePro-CalB-FWD / 5‘-GATATCTTGCCATCTGGTTCTGAC-3‘
PrePo-CalB-REV / 5‘-TCTCTTGACCAATGGGGTAG-3‘
lacZ Fwd / 5‘- GAAGGCATCGTTACAGAAGG-3‘
lacZ Rev / 5’- CTGGTGGTGGATTGCAACTCTAC-3’
CaGFP FW HindIII / 5´-TTAAGCTTATGTCTAAAGGTGAAGAATTATTC-3´
CaGFP+Stop RV SphI / 5´-GGGCATGCTTATTTGTACAATTCATCCATAC-3´
GXS1-Fw-NarI / 5´-TTGGCGCCACAAGGGAATTTGAACAATC-3´
GXS1p-Rv-HindIII / 5´-GGAAGCTTTTTGTAATTGATATATCAG-3´
XDH1p-Fw-NarI / 5´-TTGGCGCCCTTCGTTTGCAATTGGCTG-3´
XDH1p-Rv-HindIII / 5´-GGAAGCTTAGTCAAAGCGATACACAAGC-3´
CuHIS3 Fwd KpnI / 5´-TTGGTACCATGGCTGAAACGAACGGGG-3´
CuHIS3 +stop Rev KpnI / 5´-GGGGTACCCTAGAACAAAACACCCTTTG-3´
Sonde CuHIS3 Rev / 5´-CAGTGAGCGCAACGCAATTAATG-3´
RT-CuACT1 Fw / 5´-CATTGCTCCACCAGAGAGAA-3´
RT-CuACT1 Rv / 5´-GTTGGAAAGTGGACAAGGAAG-3´
RT-CuGXS1 Fw / 5´-TATGCCACATACTCCAAGGC-3´
RT-CuGXS1 Rv / 5´-AACCTGAGGCTTGTCAAAGG-3´
RT-CuGXF1 Fw / 5’-CGGTGTTAAGGCATGGAAGT-3´
RT-CuGXF1 Rv / 5’-TTCCACCTCCTCGGACTTAG-3´
RT-CuXDH1 Fw / 5’-CAAGGTCAACATCGAGCCTA-3´
RT-CuXDH1 Rv / 5’-CCATTGTGCAGGTTGAAGTC-3´
RT-CaGFPFw / 5‘-CCAGACAACCATTACTTATCCACT-3´
RT-CaGFPRv / 5‘-GCAGTAACAAATTCTAACAAGACCA-3´

Table S2. Plasmids

Plasmid / Description / Source
p3HA-URA3 / Contains 3×HA-URA3 cassette / Prill et al. (2005)
pFC1 / Contains CaACT1p-sat1-CaACT1t cassette / Setiadi et al. (2006)
pARS/AatII / Contains CaARS2 chromosomal replicator / Sonneborn et al. (1999)
pAU36 / Contains CaACT1p-StlacZ fusion / Uhl and Johnson (2001)
pGFP-URA3 / Contains GFP-URA3 tagging cassette / Gerami-Nejad et al. (2001)
pSat1-3HA / 3×HA-CaADH1t, CaACT1p-sat1-CaACT1t / This study
pSat1-3HA-ARS / As pSat1-3HA but containing CaARS / This study
pSat1-3HA-ARS-CalB / As pSat1-3HA but containing CALB-3×HA / This study
pMC1 / As pSat1-3HA-ARS but containing CuTDH3 promoter / This study
pGeneArt-CalB / Contains codon-optimized CALB / This study
pCB1 / As pSat1-3HA-ARS-CalB but containing TDH3p-CALB*-3×HA / This study
pCB2 / As pCB5 but containing TDH3p-GAS1 / This study
pCB3 / As pCB2 but containing TDH3p-gas1Δ (internal deletion) / This study
pCB4 / As pCB3 but containing TDH3p-gas1Δ-CALB*-3×HA / This study
pCB5 / As pCB1 but TDH3p-CALB*-His6 / This study
pCB7 / As pCB3 but containing TDH3p-gas1Δ-StlacZ / This study
pCB8 / As pCB1 but containing EcoRV site downstream of sequence encoding CalB pre-pro sequence / This study
pCB9 / As pCB8 but containing sequence encoding CuInv1 signal sequence instead CalB pre-pro sequence / This study
pMC2 / As pMC1 but containing TDH3p-GFP / This study
pMC3 / As pSatT1-3HA-ARS but containing CuGXS1p / This study
pMC4 / As pMC3 but containing GXS1p-GFP / This study
pMC5 / As pMC3 but containing GXS1p-CALB*-3×HA / This study
pMC6 / As pMC2 but containing CuHIS3 / This study
pMC7 / As pSat1-3HA-ARS but containing CuXDH1p / This study
pMC8 / As pMC7 but containing XDH1p-GFP / This study
pPICZ-A / P. pastoris expression vector containing AOX1p and BLE (zeocin resistance) / Life Technologies GmbH, Darmstadt, Germany
pPICZ-CalB / As pPICZ-A but containing AOX1p-CALB*-myc-6×His / This study