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4.7. Purification of recombinant protein
A cost-effective two-step purification prolocol
\\.;IS
uaerl to purify the
recombinant LTP in which, initial fr;lc~ion:ll precipit:~ti~,n uxing ammonium sulphate
frnclionation w ; ~ s followed by dialysis and gel pcrmc:~tir>n chrom:1togr:1phy. In or~lcr
to eliminate the contaminating bacterial proteins th:ll co-purify with the recombinant
protein, the supernatant after sonication kind centrifi~gati~ln was fraclloniited using
50%
ammonium sulphate. The precipitate was dissolved in phosphate huffered saline
(PBS) and dialyzed using PBS huffer (pH 7.2). The dialyzed protein samples were
slze fractionated by Sephadex G-75 column using the same hul'fcr. The fract~ona
containing fusion protein was p i e d (Fig. 15). The protein was filnher concentrated
hy lyophilization. The yield of purified LTP fusion protein w u 20 tngl.
4.8. Determination of protein solubility a n d Factor Xa cieavnge n s ~ y
When Wilkinvon and Harrison model (1991) for theoretical calculation was
employed, wheat Lrp 3FI showed 82.7% chance of insolubility when overexpressed
in
E.
coli. Therefore, pMAL-p2x expression system wllr used to enhance the
solubility of LTP fusion protein. Purified fusion protein was subjected to Factor Xa
cleavage and the cleavage products were analyzed by SDS-PAGE. Complete cleavage
of the fusion protein was observed after 6 h digestion at 4
'C,
with Factor
Xa.
However, after complete digestion, no protein appeared with an eleclrophoretic
mobility corresponding to the 11 kDa LTP. This was due to a rapid proleolysis of
released protein probably caused by bacterial protease activity that co-purify with the
LTP fusion protein.
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