3.3 Construction of α-L-rhamnosidase r-Rha1 mutants
Site-directed mutagenesis to replace the specific residue as identified
above was achieved by the KOD-Plus-Mutagenesis Kit (Toyobo, Japan) using
the specific primer pairs and the
DH5α/19-9k
plasmid as a template for polymerase chain reaction (PCR) [42]. Then
the product of PCR was transformed into freshly prepared E. coliDH5α by thermal shock at 42ºC for 90 s. The cell suspension was
cultivated at 37 °C in luria-bertani medium containing 100 μg/mL
ampicillin.
The genes were transformed into P. pastoris SMD1168 cells for
protein expression. E. coli transformants were pooled, and
the plasmid DNA was extracted using a
TIANprep Mini Plasmid Kit (Tiangen, Beijing, China). The resulting
recombinant vectors were separately linearized with Sal I, and
transformed into P. pastoris SMD1168 using a Gene Pulser
Apparatus
(Bio-Rad,
Hercules, USA). The transformants were scanned after growing the clones
on minimal dextrose medium plates at 30ºC for 3 d. Transformed yeast
cells were selected on yeast extract
peptone dextrose agar-plates containing 2.5 mg/mL G418 (Transgen
Biotech, China). Colony PCR was carried out to confirm that whether or
not the α-L-rhamnosidase gene has integrated into the genome. The
General (5’AOX, 3’AOX) and Specified primers (Q9K-F, Q9K-R) as shown
Table S3 were used for the selection of clones.