Biomedical Engineering Reference
In-Depth Information
expression in this expression system. The secretion signals were constructed
by tailed PCR, in which the tail of the PCR primer contained the secretion
signal, as well as the necessary restriction enzyme sites for direct replacement
of the trans-membrane domains with the secretion signal. These restriction
enzyme sites also facilitated the fusion of the coding regions with the
alcA
promoter. The
alcA
promoter system is an inducible promoter which can be
manipulated by controlling culture growth media conditions. This system
allowed us to mitigate against possible toxic effects of the recombinant
mannosidase protein expression, by allowing the accumulation of fungal
biomass under repressed conditions, followed by protein expression under
inducing conditions (Hintz and Lagosky 1993).
The α-mannosidase IB and IC expression cassettes were introduced
into the genome of
A. nidulans.
To confi rm secretion of active recombinant
α-1,2-mannosidase IB or IC into the culture media of putative transformants,
mannosidase activity assays were performed. After 60-65 hours of
incubation, aliquots of culture media were assayed for elevated α-1,2-
mannosidase activity compared to the non-transformed strain. Assays
of culture fi ltrates revealed several transformants with secreted α-1,2-
mannosidase activity signifi cantly higher than non-transformed strains
(which had no detectable activity) and these transformants were utilized
for further purifi cation of these enzymes.
Although crude extracellular fi ltrates from non-transformed strains
contained little to no endogenous α-1,2-mannosidase activity, it was still
necessary to further purify the expressed recombinant enzymes from
highly expressing strains for further biochemical characterization. The
α-1,2-mannosidase IB and IC proteins were thus purifi ed from highly
expressing strains, using a multi-step protocol. In each case, crude culture
fi ltrate proteins were precipitated by the stepwise increase in concentrations
of ammonium sulfate. The mannosidase activity was retained in the
40-70% portion. These proteins were isolated using a combination of gel
permeation chromatography and anion exchange chromatography. The
α-1,2-mannosidase IB was purifi ed to a specifi c activity of 7.52 U/mg, as
assayed using non-limiting amounts of the synthetic substrate Man-α-
1,2-Man-OCH
3
, while the α-1,2-mannosidase IC was purifi ed to a specifi c
activity of 4.56 U/µg. This is comparable to the specifi c activity of the
purifi ed
T. reesei
enzyme (Maras et al. 2000). Enzyme purity was confi rmed
by SDS-PAGE followed by Coomassie Blue staining (not shown). The
molecular mass of the proteins were determined by MALDI-TOF mass
spectrometry. The α-1,2-mannosidase IB had a molecular mass of ~ 60 kD
while the α-1,2-mannosidase IC had a molecular mass of ~ 63 kD.
The optimal pH for highest activity for each of these enzymes was
determined to be 5.0. Activity dropped considerably at more than one pH
