Materials and Methods
3.7 Expression of plr in M15
pQEPLR was also successfully constructed and transferred into M15 cells. The
same conduction conditions were tested as mentioned at 2.5, and three IPTG
concentrations (0.01 mM, 0.05 mM, and 0.10 mM) were tested. Nothing was added at
induction time point for uninduced control. SDS-PAGE of soluble protein fractions did
not show difference between uninduced samples and the induced samples (Fig. 25). It
Meanwhile, SDS-PAGE analysis of insoluble protein fractions showed an obvious band
at 39 KDa, as well the recombinant SDH, because of a six-histidine tag and a factor Xa
recognition site resulting in a 4 kDa increase in the molecular weight of the expressed
recombinant protein. Thus, the actual molecular weight of PLR was 35 kDa,
corresponding to the expected value. It suggests that recombinant PLR was produced in
M15 in the form of inclusion bodies. We further used Western blotting to examine the
expression of recombinant PLR in E. coli. At the soluble protein fraction, it was
surprising to find that rPLR was expressed even before IPTG induction. The signal in
the SDS PAGE was very low at the beginning, but after 6 h of induction the expression
level raised to a higher level and then kept constantly. It was also observed for both
soluble and insoluble protein fractions of uninduced control. Meanwhile, M15
transformants inducted with 0.01 mM IPTG also had an expression pattern similar to
the uninduced control in Western blotting. For both soluble and insoluble protein
fractions, the expression level obviously increased from 6 h to 15 h of induction. In
addition, M15 transformants inducted with 0.01 mM, 0.05 mM and 0.1 mM IPTG
showed the similar expression pattern in Western blotting assays, no matter in the
soluble or insoluble protein fraction (Fig. 26).
Western blotting showed that the insoluble protein fractions had the similar
expression patterns with soluble protein fractions. And obvious smear bands appeared at
the later stage (12th and 15th h). This phenomenon reveals that degradation of
recombinant PLR might happen. Although the expression in the insoluble protein
fraction was higher than in the soluble protein fraction, to avoid to get degraded and
non-function proteins, we still chose the soluble protein fraction as the materials for
enzyme reaction. The expression conditions of a minimum IPTG concentration
(0.01mM) and 9-h induction were decided.
Chapter IV
Disscussion
Two genes (plr and sdh) were cloned from Podophyllum pleianthum Hance and
were expressed in an E. coli system for enzyme’s characterization. SDH of
Podophyllum pleianthum Hance showed a high nucleotide sequence similarity and
amino acid sequence identity with the relative gene of Podophyllum peltatum (Fig. 13
and 14). Meanwhile, PLR of Podophyllum pleianthum Hance showed high nucleotide
sequence similarity and amino acid sequence identity with the relative gene of Forsythia
intermedia (Fig. 22 and 23). The conserved sequence “GxGGxG” of NAD binding
domain of SDH and “GxxGxxG” of the NADPH binding domain of PLR indicate they
can bind with the cofactor NAD and NADPH, respectively. PLR and SDH are the
corresponding enzymes involved in oxidation-reduction reaction.
In expression of rSDH, the conduction of three IPTG concentrations didn’t show
obvious difference in expression efficiency. We finally chose the minimum IPTG
concentration because of the economy and to lower the toxicity to hosts (Fig. 15). In
vitro reaction revealed that the recombinant SDH catalyzed secoisolarciresinol to
matairesinol (Fig. 18), and the conversion rate was 50% after 2 h of incubation.
According to the previous report, rSDH showed stereospecificity to its substrate (Xia et
al., 2001). In this study racemic secoisolariciresinol was chosen as the substrate, and our
results suggest the rSDH also show a stereospecificity to the substrate. We speculate
that the conversion rate could reach to 100% if we use the right substrate (+) - or
(-)-secoisolariciresinol). Kinetic parameters of rSDH were also measured. The rSDH
showed Km values ~ 231.48 μM with apparent maximum velocities ~13.25 (expressed
as μmol/min/mg protein) obtained from Lineweaver-Burk plots. Compared with SDH’s
gene from Podophyllum peltatum (Xia et al., 2001), the Km values of the SDH was
160.2 μM, and the maximum velocities was 7.1 (expressed as μmol/min/mg protein).
The values of Km and the maximum velocities didn’t express a big variation.
In expression of rPLR, it was found that PLR was to be expressed even without
IPTG induction (Fig. 24 and 25), which showed the phenomenon of leakage. And the
amount of expressed recombinant PLR was detected in the fraction of insoluble proteins.
M15 cells had a higher intracellular LacI protein concentration due to possessing
pREP4 vector. This reduces the T5 promoter activity more aggressively than the JM109
transgenic strain. There was still a difference in induction ability between 0.01 mM and
0.1 mM IPTG. Induction with 0.1 mM IPTG had a faster accumulation rate of rPLR in
the insoluble protein fraction, while induction with 0.01 mM IPTG could accumulate to
a higher level at 9 h of induction. M15 transgenic cells were incubated at 37oC to raise
cell number and inducted at 25oC for foreign protein expression. Because of the leakage
in expression, whether 37oC was still an optimum incubation temperature of increasing
cell number therefore was doubted. The expression of rPLR in the insoluble protein
fraction in E.coli suggests that the expressed rPLR was major in the form of inclusion
bodied. This phenomenon would be resolved by lowering temperature during IPTG
induction. As previous reports mentioned, PLR from Linum album (PLR-La1),
Forsythia intermedia (PLR-Fi1), Thuja plicata 2 (PLRTp2) and Linum corymbulosum
Reichenb were specific for (+)-pinoresinol (Dinkova-Kostova et al., 1996; Fujita et al.,
1999; von Heimendahl et al., 2005; Bayindir et al., 2008; Nakatsubo et al., 2008), and
PLR from Linum usitatissimum (PLR-Lu1), Thuja plicata 1 (PLR-Tp1), Arabidopsis
thaliana (AtPrR2) were specific to (-)-pinoresinol (Dinkova-Kostova et al., 1996; Fujita et al., 1999; von Heimendahl et al., 2005; Bayindir et al., 2008; Nakatsubo et al., 2008).
To prove the rPLR's function of P. pleianthum, in vitro reaction of bioconversion should
be investigated further.