The expression vector consists of MBP preceded by methionine, with the final four amino acids replaced by 23 residues encoded by the pMAL-c2
polylinker. The MBP protein was purified with affinity chromatography on amylose resin, as described in previous reports [7].
It includes different site of integration between interest gene and Pichia genome, variability of different expression host cell to secrete and express the recombinant enzyme, and different sites in the
polylinker for inserting foreign genes [31].
PCR products were digested with HindIII and EcoRV and cloned into the
polylinker region of the mammalian cell expression vector pcDNA3 (Invitrogen, San Diego, CA).
Subsequent sequencing of the 3' end of the insert and the flanking genomic DNA indicated that the
polylinker sequence containing a HindIII site was truncated by 10 bp.
-- Plasmids pO.380, a vector which contains 3.8 kb of Pu DNA subcloned into the EcoRI restriction site within the
polylinker region of pBluescribe (Stratagene) (McLean et al.
This 812-bp linked sequence was cloned into the
polylinker region of the pSP64polyA plasmid.
The
polylinker sites permitted directional cloning into single-stranded M13 bacteriophages (Medlin et al., 1988) for the dideoxynucleotide-mediated chain-termination sequencing protocol (Sanger et al., 1977).
The forward primer for vector amplification (57-CACCACCACCACCACCACTGAGAT-37) matched to the 3' side of the
polylinker region of expression vector pET28a.