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1. Factors that influence Gene Expression
|2. Expression vectors|
|5. Protein stability|
|6. How to address the hopeless case|
"Before discussing any subtleties of protein folding, such as possible constraints on the protein sequence, potential specificities of folding modulators, or the regulation of the chaperone machinery, a few global factors must be clarified.
Successful protein folding requires, of course, that the end product is a thermodynamically stable entity. Many reports of only 'insoluble material' being produced upon expression of a particular protein in E. coli are not in the least surprising, in that something thermodynamically impossible was attempted. Almost invariably, truncated domains are severely destabilized and often totally unable to fold to monomeric protein; for example, a beta-barrel from which, say, two strands are missing (e.g. because the domain was 'defined' as being located between conveniently spaced restriction sites) will generally not choose to go to the native state of the original protein. Similarly, dimeric complexes or multi-subunit assemblies may not tolerate the absence of a subunit, by virtue of a large hydrophobic subunit interface becoming exposed. In such cases, aggregation may be the only option for the protein to cover its hydrophobic surface.
Furthermore, many heterologous proteins of interest are naturally secreted and contain disulfide bonds. The formation of these disulfides is often crucial for structure formation (i.e. for the stability of an intermediate) or at least for obtaining a minimal stability in the final product. In cases such as these, expression of functional protein in the cytoplasm of E. coli has very little chance of success, unless measures are taken that favour disulfide bond formation in this compartment, even though soluble, albeit inactive, protein is sometimes formed."
cited from: J Gerard Wall & Andreas Plueckthun 1995. Current Opinion in Biotechnology 6:507-516.
Thus, the main problems for production of soluble proteins are aggregation and proteolysis during or after folding or after cell disruption.
- Inclusion bodies
Are easy to separate, can be solublized in 6 M urea or 8 M guanidinium hydrochloride. Proteins can be refolded by dilution of denaturant.
Inclusion body formation can often be reduced by growing cells at 20oC (Note that 28oC does not work nearly as well). If this is not successful, try adding 6% ethanol to rich medium when adding inducer. EtOH induces heat shock response which overexpresses chaperones and proteases. The latter are barely active at 20oC.
- Native state
Folding is often catalysed by either molecular chaperones or folding catalysts such as disulfide oxidases or disulfide isomerases and proline isomerases. Co-overexpression of those can be helpful.
- If the target protein is part of a hetero-oligomeric complex - always coexpress the other members of the complex. Complex formation is usually accompanied by conformational changes often leading to increased resistance to cellular proteases and solubility.
- Posttranslational Modifications:
Glycosylation does not yet work in E. coli
Disulfide bonds do usually not form in the cytoplasm. Disulfide bond formation is catalysed and monitored in the periplasm. In the cytoplasm, S-S bonds can form spontaneously in trxB, gsh, gor mutants, trxB gor double mutants being the strongest combination. Isomerisation of S-S bonds can be promoted by coexpression of signal sequenceless disulfide isomerase (dsbC).