Cloning and expression of an active aspartic proteinase from Mucor circinelloides in Pichia pastoris

BMC Microbiology

Table 2 Oligonucleotides used for PCR amplification in this study

Primer name	Sequence (5′-3′)
Primers for DNA fragment PCR to obtain a partial sequence of genomic DNA of the acid proteinase
12 ND-F	AACGATATCGAGTACTATGGT
M.cir-2R	TTAAAGACTTCATAGTTGTTCTT
Primer for 3′-RACE PCR (Gene-specific primer)
GSP-Mucor-1 F	GATGGTCGTGCCTGGTCTATCCAAT
Primer for 5′-RACE PCR (gene-specific primer)
GSP-Mucor-2R	CATTGTCTCTGGCACCGTATTGAGCAGC
Primers for full-length cDNA and recombinant plasmids
APMC-EcoNaeI-F	ATGGAATTCGCCGGCGCTACTACTGATGCCACTGGTACTGTCCCCG
APMC-F	AGGAATTCTTCTCATTAGTCTCTTCTTG
APMC-Met-F	ATGGAATTCATGAAATTCTCATTAGTCTCTTCTTGTGTC
MCAP-3 F	TATCTCGAGaaaagaGCTCCCAGTGGTAGCAAGAA
XhoI-N-MCAP-F	TATCTCGAGaaaagaATGAAATTCTCATTAGTCTCTTCTTGTG
APMC-NotI-R	AAAGCGGCCGCGACAGATTTGGCAATTT
APMC-Stop-R	GTGATTTATAGATAGATAGATGAAATGTACCAAA
Primers to identify clones containing recombinant plasmids
pGAP-F	GTCCCTATTTCAATCAATTGAACAAC
AOX1pGAP-Rev	CAAATGGCATTCTGACATCCTC

The underlined sequences (GAATTC; EcoRI, CTCGAG; XhoI and GCGGCCGC; NotI) represent the additional restriction sites at the 5′ ends of forward and reverse primers. The lowercase letters indicate the Kex2 cleavage sites. The primers (for First-strand cDNA synthesis, 3′-RACE cDNA and 5′-RACE cDNA) provided in the SMART RACE cDNA Amplification Kit (Clontech) are not described in the table.

ISSN: 1471-2180