> I have been trying to express a rat protein in bacteria. The MBP-fusion > expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag > only gave inclusion bodies. The problem is that all protein runs in the void > volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl), no > matter it is the intact MBP-fusion or cleaved sample. There is no Cys on > this protein so there is unlikely any disulfide bond related problem. > Anything I can do before I throw away this construct and try insect or > mammalian cells? Thanks. >
First of all, using a carrying protein (like GST, MBP) can be disconcerting. These proteins are very soluble and can solubilize an insoluble protein in testing condition. So you have something soluble but your protein of interest can be misfolded or can precipitate when the carrying protein was cleaved. So keep in mind that a soluble carried protein is not always a good protein. After this consideration you have a wide range of conditions to test. - Solubility of your MBP-fusion: ultracentrifugation, thermal shift assay (in different pH, salt, salt concentration), micro-dialysis - Folding of your MBP-fusion: circular dichroism, 1D NMR (if you can, compare with MBP alone) - Aggregation/monodispersity of your MBP-fusion: DLS (in different pH, salt, salt concentration) - For gel filtration assays don't forget that MBP can dimerize The second part of your tests can be expression conditions. Sometimes low but native expression is better than high but carried or insoluble expression. - Medium - Temperature - Host cells (different E. coli, yeast, insect cells...) - Inducing strength - Co-expression with ligands or chaperones And the last but not the least part of your tests can be refolding. Inclusion body expression is the first step of your purification. If you have his-tagged protein in inclusion body a one step purification can be performed in denaturing conditions. A wide range of refolding conditions can be tested: - Flash dilution - Dialysis - Refolding by slow gradient on an Ni column (if you have an his tag) - pH, salt, detergent conditions - Chaperones OD at 340 nm can monitor the refolding efficiency. Good luck Michel
