signal peptide and transmembrane.. - (Jun/13/2006 )
Currently i'm working with recombinant lipase 205y genes from Bacillus sphaericus. As my objective is to crystallize the protein, thus as far as i am concerned, i have to cut the signal peptide as well as the transmembrane segments so that the segments will not interfere during the crystallization prosess. However, problems arise as the activity for both segments (without signal peptide and transmembrane segments) were very low (when observed onto tryibutyrin plates and lipase assay) when compared to the full sequence genes (open reading frame). Thus, i cannot proceed my work as i'm expecting a funtional enzymes with high activity. I'm using pUC19 and pBAD as the cloning and expression vectors, respectively. I used to cloned both genes (without signal peptide and transmembrane segments) into pET-100, pQE-30UA and pET-22b(+), however, the results remain the same. Thus, the issue are;
1. Shall i proceed with purification of the enzymes regardless the presence of signal peptide and transmembrane segments in the genes?
2. Is it possible for me to crystallize the protein even with the presence of signal peptide and transmembrane segments?
3. Does the vectors that i used seems to contribute to the low activity of the enzymes (maybe not compatible enough)?? Should i try other vectors..e.g. pGEX?
Please give some comments and suggestions. Thanks a lot!
Normally the signal peptide would be cleaved in the active form in the native environment. Do you know if this is true? If so, it is unlikely that the removal of the signal peptide woula affect the activitity (except in a positive way, perhaps). The problem is likely the transmembrane region. Is this at one end of the protein? You might try keeping the part nearest the remainder -- there might be an interaction. You could also try preparing the full length one to verify activity and quantitate the loss of activity when it is removed.