The protein sequence/structure, as a whole, is optimized thanks to the evolutionary machine. It is quite common to have different expression profiles for mutants-deletions-variants of the desired protein.

The result of the expression of your construct is purely empirical. The small domain you deleted could have affected the folding and/or the solubility of your protein (e.g. a small helical bundle of the RuvA protein that is required for the solubility of the entire protein). Keep in mind that sometimes you can even reach higher expression, but it all depends on the protein and on the truncation.

For example you can try different constructs, or replacing the deletion with a small insertion. Don't be greedy with your ligase .  

For a general review on how to detect good boundaries for your protein domains see:

http://www.ncbi.nlm.nih.gov/pubmed/20658975

The protein sequence/structure, as a whole, is optimized thanks to the evolutionary machine. It is quite common to have different expression profiles for mutants-deletions-variants of the desired protein.

The result of the expression of your construct is purely empirical. The small domain you deleted could have affected the folding and/or the solubility of your protein (e.g. a small helical bundle of the RuvA protein that is required for the solubility of the entire protein). Keep in mind that sometimes you can even reach higher expression, but it all depends on the protein and on the truncation.

For example you can try different constructs, or replacing the deletion with a small insertion. Don't be greedy with your ligase .  

For a general review on how to detect good boundaries for your protein domains see:

http://www.ncbi.nlm.nih.gov/pubmed/20658975

Proteins evolve as combination of domain. Many servers help in designing truncations of proteins. I find ProteinCCD particularly useful.

ProteinCCD (CCD for Crystallographic Construct Design) aims to facilitate a common practice in structural biology, namely the design of several truncation constructs of the protein under investigation, based on experimental data or on sequence analysis tools. ProteinCCD functions as a meta-server, available online at http://xtal.nki.nl/ccd, that collects information from prediction servers concerning secondary structure, disorder, coiled coils, transmembrane segments, domains and domain linkers.

For an explanation, see: Moojg et al, NAR, 2009

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703965/

Hi !

Thanks a lot for good answer

If the structure of your protein is known, or your protein is homologous to a known structure, that can be an essential guide to determine whether the "small domain" is required for the overall fold, or is dispensable. There may be interactions between domains that will leave hydrophobic patches that cause aggregation if one domain is deleted.

One good technique to determine optimal sites in linkers or loops suitable for cleavage is limited proteolysis followed by mass spectrometric analysis. An array of different proteases that cleave at different residues can be used to define optimal cut sites. For structural biology, the optimal protease and concentration can be applied via in situ proteolysis, or you can make a new clone with the optimized boundaries. Some publications that address this technique are:

http://www.ncbi.nlm.nih.gov/pubmed/16211509

http://www.ncbi.nlm.nih.gov/pubmed/20685133

thank you for answers, if this domain affect folding of protein, can it cause the expression decreased?? protein can be expressed as inclusion body. how does solubility affect expression?

Sure, especially if the domain is important for folding or for maintaining the 3D structure of the protein, trying to delete it could make it impossible to purify any protein. It would be helpful to know what the protein is. Is there a reason you need to delete this domain?Expression is affected by so many things. You may need to try another (non-bacterial) expression system (insect cells, yeast, mammalian cells). Or, if you delete the domain, you may need to make compensatory mutations to account for the loss of that domain. Is the protein normally glycosylated? If so, that could affect expression and solubility if you're making it in E. coli. Does it have disulfide bonds? Is it a membrane protein, or membrane-associated (does it have any predicted transmembrane helices?) If so, you likely need detergent to purify it from inclusion bodies.

hello Dear Micheal

my protein is enzyme, cellulase. I delete small domain called Ig-like. Molecular simulations show this domain has function in stability of protein and deletion of this protein cause  enzyme loses its activity. this enzyme has been expressed experimentally without Ig-like . it has activity, but its expression decreased.

my reason for deleting this domain is to understand function of this domain.