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Table 1 Microorganisms and plasmids used in this study

From: Identification and characterization of N-glycosylation site on a Mucor circinelloides aspartic protease expressed in Pichia pastoris: effect on secretion, activity and thermo-stability

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
  1. aThe insert was cloned flush with the Kex2 cleavage site and in a frame of the native signal sequence and in a frame with C-terminal polyhistidine tag into the XhoI and NotI site of the pGAM1
  2. bThe insert was cloned downstream of the GAP promoter and in a frame with the C-terminal polyhistidine tag into the XhoI and NotI site of the pGAM1