Does single nucleotide mutation in Ribosomal Binding Site of an expression vector hamper protein expression? As there is no band could be seen in SDS-PAGE..
Mutations in the RBS would certainly influence the rate of translation, but to what extent depends on the exact mutation and the context of that mutation. For recombinant protein expression, the lack of expression could be due to many factors. Some proteins simply do not express; and in some cases a single amino acid difference can make a huge difference in the level of expression of a protein. I think you may want to try different culture/induction conditions, for example, at lower temperature (18C) or auto-induction. Also you may want to try different expression vectors, or as a fusion protein to MBP or GST, etc.
Mutation in ribosomal binding site may significantly influence the process of translation, or just decrease the rate of translation. Expression failure can be caused by a lot of reasons. You would need to repeat the experiment, optimize the expression conditions, or design a new experiment to determine the cause. This picture (is taken from www.biologicscorp.com) lists some common causes of failure in E. coli and hopes to help.
thanks Alison Rong , Ali Mahmoudpour & Hua Xiao for your valuable suggestions..
The iGEM website has a list of RBS sequences that are recommended for protein expression in E. coli. You could at least compare your RBS sequence to these. Of course, as discussed above, there are many other factors that can affect protein expression.
http://parts.igem.org/Ribosome_Binding_Sites/Catalog
Elise Rivett thanks for the suggestion... I will definitely take this into consideration..
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