Generally, people can use either deletion or overexpression strategy to do characterization of any novel protein candidate. Can anyone describe or suggest any reference paper discussing the pros and cons of the above mention strategies?
Hi, Md Atikur Rahman As I understood your question, you need to characterize a novel protein. I might suggest three ways to do it, depending on the functions of your protein.
1. If it is a signaling ligand such as cytokines (IL-6, TNF a) you can clone it into a vector to make recombinant proteins with a fusion protein tag and treat the protein into cells. Then you can study the downstream signaling and cellular events such as apoptosis, proliferation, or inflammation.
2. If it is a transcription factor such as IRFs, you can clone it pcDNA3.1(+) vector and you can transfect into cells using a transfection reagent. Then you can study the effect of overexpression of your protein.
3. if it is some signaling intermediate or adaptor protein, you can do both overexpression or knockdown using siRNA or another method. Please refer to our publications. We have summarized the vectors and protocols in our papers (IL-11 for rec. protein purification, CateninB, and IRF 4 papers for overexpression and knockdown protocols. Good luck with your future endeavors.....
I think deletion/knockdown is more useful to know the function of unknown protein. Overexpression with a reporter would be useful to know localization of the protein but it won't tell what the protein is exactly doing there.
As noted in previous comments, deletion/knockdown facilitates protein function identification, while overexpression favors localization. In addition, the overexpression of heterologous recombinant proteins is helpful for the characterization of the biophysical properties of the protein, and combined with site-directed mutagenesis, it can also be applied to the study of protein interactions and the determination of enzymatic active sites.
The knock-out is the standard, both knock-out and over-expressor are better. However, the difference is in number of homologues (if you have 3+ genes with the same function, you probably won't see any phenotype in low-order mutants). Also, with with overexpressor, you don't need to select homozygot, so it's faster. Additionally, you can prepare inducible-overexpressor or tissue- or process-specific promoters, so that you can study specific changes directly upon increased production of your protein or influencing specific process.
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