Essay On Folding Analysis Of Protein
3.2 Folding analysis of recombinant TGF О І1 protein
To analyze the folding of recombinant TGF О І1, purified protein was electrophoresed
on native gel, under non reducing condition and stained with the silver stain.
Electrophoresis of the purified TGF О І1, under non reducing condition, confirmed
the presence of dimeric form of the TGF О І1, as a 25 kDa band on the non
denaturing gel (Figure 5 ). Here, there was no trace of a monomeric TGF О І1 band
(13.5 kDa). This result indicated that the mature form of TGF О І1 is probably folded
properly, and secreted as a dimer into the culture medium.
3.4 Biological activity of recombinant TGF О І1
The biological activity of purified TGF О І1 was measured in classical growth
inhibition assay on Mv1 Lu cells. ... Show more content on Helpwriting.net ...
Complicated processes of synthesis, folding and activation cause difficulties to
achieve active TGF О І1 from expression systems. Most previous studies were
performed to express the full length of TGF О І1 in mammalian cells and baculovirus
insect expression systems [11, 21, 22]. However, as the major secreted proteins by
theses cells, consisted of non covalently associated propeptide with the mature form
of TGF О І1, the yields of biologically active purified proteins were not sufficient,
and significantly decreased after removal of LAP propeptide [11]. Therefore, some
attempts were made to express biologically active TGF О І1, using the DNA
sequence, encoding the mature domain in eukaryotic and prokaryotic systems.
However, the expressed proteins were not active, and in some cases, chemical
refolding was required, to maintain the biological activity [9, 10]. Here, we showed
that the expression of biologically active TGF О І1, without the requirement of LAP
coding sequence and chemical refolding, is feasible in yeast expression system. In
addition, one limitation for the accumulation of mature TGF О І1 in the reported
expression systems, was the low stability of the expressed protein, resulted from the
activity of endoproteases. To overcome this problem, we used the engineered
PichiaPink, strain 3, knocked out for protease B. Similar to higher eukaryotes,
secreted proteins in yeast can be processed