PENGEKSPRESAN PROTEIN LAKlJRAN prM BERBIOTIN UN11JK \t1R.US JAPANESE ENSEF ALITIS (JE) DAN VIRUS DENGGI 2 DALA.\1 E. coli
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Date
1995-04
Authors
LEE, WONG HUI
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Abstract
This project attempted to produce biotinylated fusion protein of prM gene
belonging to 2 flavivirus, Dengue 2 and Japanese encephalitis (JE) using the Pinpoint Xa ยท
3 expression vector. Two clones, pl45 (PPXa3-JEprM) and p133 (PPXa-Den2prM) were
transformed into XLIB!ue E. coli and PCR-screened for insert. Fusion protein expression
was checked by Western blots. The sized proteins were probed by streptavidin-HRP
(1 :3000) and polysera with antibodies recognizing prM. PPXa3-JeprM without signal
peptide was constructed from p145, transformed into XLI Blue (p149) and ToplOF'
(p150) and protein expression checked on Western blot. No fusion protein was detected
for t~e 8 positive clones of p145 and p133 each, 7 p149 positive clones and 9 p150
positive clones. The PPXa3 leaky expression is suspected to aggravate the fusion
protein's toxicity. Attempts to control the leaky expression by manipulating the
fermentation's parameters didn't increase the amount of the four fusion proteins to a
-,
detectable level on Western blot. The presence of the signal peptide in the JE construct and
E.coli intraspesies genetic variability didn't show a significant difference in the protein
expression patterns.
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PENGEKSPRESAN PROTEIN LAKlJRAN prM BERBIOTIN UN11JK \t1R.US