Posts Tagged ‘Functional annotation of hypothetical proteins’

Functional Annotation of Hypothetical proteins

April 30, 2014 Leave a comment

Cloning, expression and purification of difficult to clone, express and purify proteins in E. coli 

Experimental work is though time taking but direct approach for functional annotation of hypothetical proteins; however, at times it is difficult to decide upon the experimental design for a relatively new class of a protein. With increasing size and quality of various protein databases, it is becoming relatively easier to look for the experimental design for the probable function of a protein. Following are the steps that can be used in choosing the type of experimental analysis that needs to be performed and the substrate to be used during laboratory tests.

If the protein is predicted to be an enzyme, BLAST results normally indicates its closely related proteins that can be looked upon for the experimental procedures to be performed as indicated by the matching hits (look for the papers on those proteins that might indicate the type of related function the protein might perform).

With the increasing domain databases, it is possible to analyze the protein domain wise indicating the ability to perform certain kind of biochemical reactions if any. The NCBI’s Conserved Doamin Database (CDD), Pfam and InterProScan databases have a large number of conserved domains that defines a functional class. Presence of certain domain is also indicative of the possible activity of the protein and therefore the type of substrate to be used for defining its chemical activity in laboratory could be helpful.


Composition based analysis of protein: there are various bioinformatics tools available online to studying the amino acid composition based analysis of protein informing various properties which help in indicating the properties of protein which later help with the functional annotation of the proteins i.e. ProtparamSPAANMP3 and a lot more etc.

Homology based modeling: this is an important step in determining the functional annotation of protein based on the structure of the protein, though it may be difficult for the proteins with low identity (<30%) with the already known crystal structures of the protein. However, a good homology model can be an important step towards determining functional annotation for a protein. So also the secondary and tertiary structure prediction of the protein will tell the similar functional categories thereby help in designing relative experimental assays. Some of the commonly used homology based modeling tools are listed here


Phylogenetic analysis: Phylogenetic analysis not only shows evolutionary divergence of the protein but also act as an important step towards functional conservation of the protein. This helps in determining the degree of functional similarity with other related homologous proteins. Thus, determining the appropriate experimental assays towards functional annotation of the protein. With the help of molecular dynamic simulation, this also helps in-silico assessment of the ability of substrate to bind to the protein. In fact it can cut down from large number of substrate molecules to the top most hits, helping to prioritize the experimental analysis, saving time and resources.

phylogenetic analysis

It is sometimes a bit difficult while working with novel proteins for which relevant data is almost negligible worldwide, so you can wait till you get more information.

Let me know if you have more suggestions to add on.

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