Hi Nii Sama,

yes it is possible that your recombinant protein may not exhibit the required activity, due to one or more reasons. I am assuming that the protein is cloned and overexpressed in E.coli.

firstly you should check if the cloned gene ORF is in frame w.r.t. the start codon provided in expression vectors. becoz if this is the problem, then your protein will be expressed and will be seen on sds page gels and blotts but will have no activity as it is translated incorrectly.

secondly ,see whether the overexpressed protein is accumulated in Inclusion bodies or in soluble fraction. if in IBs, then make sure that the protein is extracted in soluble fraction, and purified in native conditions , as retrieving proteins from Inclusion Bodies (IB) is usually is done using urea , which completely denatures the protein , which needs to be put through re-folding conditions. this leads to loss/decrease in activity. the protein can be brought in soluble fraction by modifying the concentration of inducer eg. IPTG  0.4mM instead of 1 mM or by altering the growth temperature of culture eg. 30degrees celcius instead of 37.

also lastly see if there is some problem with the reagents or assays used to test the activity , viz. pH, molarity etc. use any positive controls if you can.

you can refer to the following link for further clarification regarding purification steps:

http://wolfson.huji.ac.il/purification/PDF/Protein_Refolding/NOVAGEN_NiNTA_purification_resins.pdf

all the best .

Prachi

Producing inactive recombinant protein is a fairly common problem. Some of the reasons for this include:

a) The protein isn't folded properly by the host expression system (e.g. crucial interactions with other proteins from the donor organism are missing, like certain chaperones for example).

b) The protein undergoes post-translational modifications in the donor organism to become active/inactive that are lacking in the host expression system (e.g bacterial expression systems like E. coli don't phosphorylate recombinant proteins).

c) The expression system accumulates the recombinant protein in inclusion bodies because it's mildly toxic to the host cells (properly toxic proteins simply kill the cells and produce little or no protein yield, which is a different problem), or it isn't soluble enough in the host cells or it aggregates into large multimers.

Sometimes you can solve these problems by simply changing expression strain (E. coli C43 instead of BL21 for membrane proteins) or expression system (e.g. try eukaryotic systems instead of prokaryotic ones for post-translationally modified proteins).

Maybe all you need is a certain co-factor that your protein needs (e.g. common metal co-factors include Mg and Zn), which is either not supplied by the expression system at the levels you require or which is stripped away during your purification protocol (e.g if you add EDTA to remove possible Ni contamination after a Ni-NTA step). In this case all you need to do is add enough of the co-factor to your protein at the end of your purification procedure.

In addition to the above answers regarding problems with folding, inclusion bodies, modification and co-factors, the problem might also be that you are just not getting expression. How have you confirmed that the protein is being expressed, as lack of expression is the most common problem. 

Thank you  Prachi Nawkarkar and  Antonio Ariza and Michael J. Benedik .

Antonio Ariza how can i add co-factors at the end of purification procedure? any protocol?

Michael J. Benedik ,the common procedure is SDS-PAGE and Western Blot tests.

Is there any other protocols? or are these two protocols wrong for the detection of protein expression? 

Nii, you mentioned your protein is the human growth hormone, which is a very well-studied protein ... I just did a quick check and I couldn't find anything regarding the need of a co-factor for this protein and that means you don't need to add any co-factors to activate it.

Out of curiosity, how are you testing its activity? From what I read it doesn't have any catalytic activity, it simply binds to a receptor in order to activate the MAPK/ERK pathway. Therefore, I'm guessing you're using some kind of binding assay and, when you say your recombinant protein is inactive, you mean it doesn't bind in the assay. Is that correct?

Thanks dear Antonio. i hope your answers will helpful.

I want to produce and purify rhGH and i asked this question to see if this problem would  happen to proteins like mine .if it does happen what should i do?after your answers and with your description about this protein,  we should use activity assays just for Enzymes or something like that and proteins like rhGH don't need activity tests.is that correct?

but what is the biological activity that some publications do on proteins like recombinant human Insulin-like growth factor (rhIGF)? like the following paper:

Article Expression and efficient purification of tag-cleaved active ...

The first thing that springs to mind is a binding assay, for which you would need to immobilise whole receptors or at the least the part of the receptor that your hormone binds to, then you add your hormone, let them interact, then you wash the system to remove unbound hormone and finally you visualise if your hormone is bound to the receptors. This is basically an ELISA, but it means you need to be able to produce and purify the receptors (or the part of the receptors the hormone binds to) and you need antibodies against your hormone. This might be difficult and expensive to achieve.

The other option I see is probably better. For this one you should test if "something" is activated by your hormone further downstream in vivo. If you add your hormone to a plate culture of a cell type that reacts to your hormone, you can test if the hormone has the desired effect (e.g. cells grow larger, proliferate, etc...). You can then compare this to control plates where you add a blank (the same volume of buffer that your hormone is in and which contains everything that your hormone sample has, except the hormone itself) or to plates of a cell type that shouldn't react to your hormone (here you add the hormone to cells that lack receptors that react with your hormone).

Another option is to check the levels of the molecules involved in the downstream cascade elicited by your hormone or if they undergo a change like phosphorylation. If your hormone is active, it should bind to the receptors of suitable cells (like in the previous experiment) and activate the MAPK/ERK pathway, which means you can check if the enzymes in this pathway change (e.g. levels of phosphorylated MAPK in treated and untreated cells).

I'm not a biochemist or cell biologist, so I don't carry out these types of experiments myself, but I'm sure you can find further help if you ask specific questions on this in the forum. In any case, I'd recommend you read up on suitable literature first.

I hope this helps.