Table 1 Microorganisms and plasmids used in this study
Strain or plasmid | Genotype | Source or references |
|---|---|---|
Escherichia coli | ||
 Top10 | F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG | Invitrogen |
Pichia pastoris | ||
 X-33 | Wild type | Invitrogen |
 SMD1168 | Protease deficient | Invitrogen |
Plasmids | ||
 pGAPZα-A | The pGAPZα-A vector uses the GAP promoter to constitutively express recombinant proteins in Pichia pastoris | Invitrogen |
 pGAM1 | A pGAPZα-A derived vector where the α-MF is replaced by mcSP and uses the GAP promoter to constitutively express recombinant proteins in Pichia pastoris | This work |
 pGAPZα + MCAP-5 | pGAPZα-A derivative carrying the MCAP gene without a signal sequence and without intronsa | Gama Salgado et al. (2013) |
 pGAM1 + MCAP-3 | pGAPZα-A + MCAP-3 derivative carrying the MCAP with mcSF without an intronb | This work |
 pGAM1 + MutMCAP-8 | pGAPZα-A + MCAP-3 derivative is carrying the MCAP without an intron. The codon of the amino acid asparagine 331(AAC) was changed to Glutamine (CAA)b | This work (Single mutant Asn331-Gln) |
 pGAM1 + SyMCAP-6 | pGAM1 + SyMCAP-6 derivative carrying the MCAP gene with native signal sequence and without intron. The original MCAP gene was adapted to the optimal codon usage of P. pastoris. The insert was cloned flush with the Kex2 cleavage site and in frame of the native signal sequence and in frame with the C-terminal polyhistidine tag into the XhoI and NotI site of the pGAM1b | Gama Salgado et al. (2013) and in this work |