What the hell is NS4B?
we start designing any expression experiments, we should gather some information!
NS4B is a non-structural protein
of the Hepatitis C virus. To get more information about it (or any other
protein), we can search Entrez (including Pubmed) or SRS using
and hepatitis C as keywords. However,
if we have a sequence of the protein, it may be shorter to use it to search
two databases of domains within proteins: Interpro
(using Interpro scan)
Luckily, our virtual boss
supplied us with a sequence (both DNA and protein),
so here are the search results:
Family and PF01001 linked entries (same
info presented in diferent ways) and the abstracts of the
linked pubmed entries.
Click on the red bar of
HCV_NS4B to get to the two pubmed links. Use the "Related articles" option
of each Pubmed entry to find additional valuable information.
Go through all
this stuff (~10-15 min),
have we learned so far?
suggest anything as to the design of the expression/purification experiments?
answers and choose between "right" and "wrong".
part of a large polyprotein precursor
that is proteolytically processed into at least 10 distinct products, in
the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B
cleaved from both sides (4A/4B, 4B/5A) by a serine proteinase which is
another part of this polyprotein precursor - NS3.
with NS4a and NS3 to form a large replicase complex to direct the viral
hydrophobic, suggesting a possible membrane-related function.
translation in-vivo (using HeLa and Cos-7 cells). [This data
was published in a few "related articles"].
individually or in the context of the entire HCV polyprotein, NS4B is localized
in the endoplasmic reticulum (ER), as shown by subcellular fractionation,
immunofluorescence analysis, and double-label confocal laser scanning microscopy.
data was published in a few "related articles"].
may be more than one "right" answer.
project is too complicated. Let's abandon it.
possible involvement of NS4B in replication and in protein synthesis may
be more crucial in eukaryotic expression systems, since the virus Hepatitis
C uses the host machinery of human cells. Using various bacterial strains
or the RTS in-vitro translation system may be better.
using the RTS in-vitro translation system, we should consider using detergents
as additives, since NS4B is hydrophobic.
should not be expressed as a fusion protein with any of the usual fusion
partners. It can only be fused to NS4A or NS5A.
you examined all the possibilities?