From: Cloning and expression of an active aspartic proteinase from Mucor circinelloides in Pichia pastoris
Strain or plasmid | Genotype | Reference |
---|---|---|
Strains | ||
Escherichia coli | ||
TOP10 | F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG | 10 |
Pichia pastoris | ||
X-33 | Wild type | Invitrogen |
Mucor circinelloides | ||
DSM 2183 | Wild type | German resource centre for biological material |
Plasmids | ||
pGAPZα-A | The pGAPZα-A vector use the GAP promoter to constitutively express recombinant proteins in Pichia pastoris. Contains the zeocin resistance gene (Sh ble). | Invitrogen |
pGAPZα+MCAP | pGAPZα-A derivative carrying the whole MCAP gene1. | This work |
pGAPZα+MCAP-2 | pGAPZα-A derivative carrying the MCAP gene without an intron1. | This work |
pGAPZα+MCAP-3 | pGAPZα-A derivative carrying the MCAP gene without an intron2. | This work |
pGAPZα+MCAP-5 | pGAPZα-A derivative carrying the MCAP gene without a signal sequence and without an intron2. | This work |
pGAPZα+MCAP SP-1 | pGAPZα-A derivative carrying from the amino acid sequence number 67 to 394 of the MCAP gene without an intron1. | This work |
pGAPZα+SyMCAP-6 | pGAPZα-A derivative carrying the MCAP gene without 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 α- factor signal sequence and in frame with the C-terminal polyhistidine tag into the XhoI and NotI site of the pGAPZα-A. | This work |