What the hell is NS4B?

Right!   Before 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 NS4B 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) and CDD.
Luckily, our virtual boss supplied us with a sequence (both DNA and protein), so here are the search results:
Interpro Results
Read  InterProIPR001490 Family and PF01001 linked entries (same info presented in diferent ways) and the abstracts of the linked pubmed entries.
CDD Results
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),

What have we learned so far?

  1. NS4B is 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 -COOH.
  2. NS4B is cleaved from both sides (4A/4B, 4B/5A) by a serine proteinase which is another part of this polyprotein precursor  - NS3.
  3. NS4B interacts with NS4a and NS3 to form a large replicase complex to direct the viral RNA replication.
  4. NS4B is hydrophobic, suggesting a possible membrane-related function.
  5. NS4B suppresses translation in-vivo (using HeLa and Cos-7 cells). [This data was published in a few "related articles"].
  6. When expressed 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. [This data was published in a few "related articles"].
Does this suggest anything as to the design of the expression/purification experiments?
Read ALL answers and choose between "right" and "wrong".
There may be more than one "right" answer.

1. This project is too complicated. Let's abandon it.

2. A 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.

3. When using the RTS in-vitro translation system, we should consider using detergents as additives, since NS4B is hydrophobic.

4. NS4B should not be expressed as a fusion protein with any of the usual fusion partners. It can only be fused to NS4A or NS5A.

Have you examined all the possibilities?