We consider expressing this
protein in bacterial strains. Thus, we should further characterize this
protein (and the nucleotides encoding it), and see if we have any indications
to choose a specific bacterial strain.
Let's run the following analysis:
Are there rare codons in the
Does the protein contain S-S
Are there any localization signals
we should consider removing?
Examine all the results (~10-15
of Current Search
Several rare codons exist within
the nucletide sequence, including three pairs of consecutive rare codons.
Cysteines 257 and 261 are predicted
to be part of S-S bonds, whereas cysteine 187 is predicted to have a free
SignalP (where this sequence
is defined as "eukaryotes") predicts a cleavage site of a
signal peptide between residues 34 and 35. (This prediction is given
based on both neural networks (NN) and hidden Markov models (HMM). This
gives more credit to this prediction.)
Psort II (where this
sequence is defined as "yeast/animal") suggests a cleavage
site between residues 34 and 35, a transmembrane domain between positions
175 - 191 and the C-terminus is recognized as a cytoplasmic tail. The overall
prediction for this protein's localization is:
17.4 %: mitochondrial
17.4 %: nuclear
%: endoplasmic reticulum
4.3 %: Golgi
4.3 %: vesicles
of secretory system
4.3 %: peroxisomal
Note that the literature
shows that NS4B is
located on the ER membrane, whereas the computational prediction for this
localization is rather low!
5. Phosphorylation may occur
on serines 88, 159, 164 (most
probable); threonine 165, tyrosines 6, 223 .
6. O-Glycosylation may occur
on no residue (according to NetOGlyc 3.0 Server),
or on serines 113, 227, 258 and threonines 165, 234
probable), 259 (according to YinOYang 1.2).
7. N-Glycosylation does
not occur (according to NetNGlyc 1.0).
suggest anything as to the design of the expression/purification experiments?
answers and choose between "right" and "wrong".
may be more than one "right" answer.
sequence should be expressed in Codon Plus (RIL) bacterial strain.
sequence should be expressed in Rosetta-gami bacterial strain.
we truncate the C-terminal of the protein, it will have a better chance
to be expressed as a soluble protein in bacteria.
sequence should be expressed in Glycoria bacterial strain, which allows
sequence will be phosphorylated on serines 159 and 164 upon expression
in any bacterial strain.
this protein contains hydrophobic segments and is predicted to have a trans-membrane
domain, we should express it in bacteria engineered to have more membranes
(C41 or C43).
repeat my previous suggestion: this project is too complicated. Let's abandon
it. (Better late than never!!) .
Have you examined all possibilities?
Based on the computational analysis,
we've decided to use the construct our collaborator sent us. This is pDEST15-NS4B,
where NS4B is fused to GST(so we expect a product of ~53 KDa). We've expressed
it in two bacterial strains: Rosetta pLysS and Origami B pLysS. As a control
we used another construct: pDEST15-GFP (a product of ~55KDa is expected).
In addition we've tried to express this protein using Roche's RTS in-vitro
translation system, which is based on E-coli extracts. Here we used the
control vector supplied by the manufacturer - pIVEX-His-GFP. (A product
of ~30KDa is expected).
If all three expression
systems fail to express NS4B, we'll consider either truncations and re-cloning
of the protein or using an alternate expression system.
First, let's examine the
profile of the Origami strain.