You can check the folding status of your protein by CD (Circular Dichroism).

If the protein is an enzyme that catalyzes a chemical reaction, it is super easy:

observe the formation of product from substrate or the depletion of substrate via absorbance or fluorescence or radioactive labeling. If any amount of product is being formed then your enzyme is folded.

If your protein is soluble that's a good sign. Try determining its stability in increasing concentrations of a denaturant such as guanidine-HCl or Urea by monitoring intrinsic tryptyophan or tyrosine fluorescence.

Dear Vikram Kumar

there are several different approaches that with different resolution and degree of reilablility can provide suggest if the recombinant protein expressed is correctly folded.

In general unfolded proteins are not stable an tends to aggregate under concentration because when the protein is unfolded its hydrofobic core, which is buried in the folded conformation is partially exposed and can induce non specific protein-protein interactions.

Therefore some methods used for determine protein stabity eg:

- Circular dicroism

- Differential scanning fluorimetry

- 1H-NMR and 15N-1H HSCQ NMR

can be used to have an idea of the protein folding and the fact that the protein do not precipitate when concentrated up to uM range (>5-10 mg/mll) is already a good sing.

OF course if you know from literature that your protein has:

- some activity that could be monitored with enzimatic assay or other activity test

- is able to bind some other proteins and its binding could be monitored by ELISA, SPR or BLI

- is contain some conformational epitope recognized from a mab

you can use alll this infromatino to design a specific assay suitable in your case.

However there are also intermediate cases:

eg the protein is expressed in a folded status (good CD or DSF spectra) but it miss a cofactor (eg a metal or a sugar) to become active. Is very important that you mix the results of the in vitro experiments with litetature to produce the best protein.

best regards

Manuele