It is said the ribosome binding site (RBS) should be 6-7 base pairs in prokaryotic systems from the start codon ATG. But in my case I have a sequence which is "AGGAGA TACCCTT CAG GGA TCC ATG" where AGGAGA is the RBS and the start codon ATG. Will this have an effect on protein expression?
It will probably have an effect on translational initiation efficiency, the geometry of ribosome is given and prokaryotic ribosome does not scan the mRNA for initiation codon. But what can also happen is initiation with different tRNA - as long as it is bound to IF2 and delivered to the ribosome.
In my experience with E.coli cell free expression system, these systems are much more sensitive to the applied template then expression in E.coli cells. I personally use pET21a vector with generally high yields (the sequence is AGGAGA TATACAT ATG). It's very easy to prepare constructs with this vector, as CATATG is a restriction site for NdeI.
And what also matters is the leader sequence prior the RBS (e.g. gene 10 5' leader sequence).
Hi
Thanks for your reply. As of now I generated two more constructs
1. "AGGAGA TACCC ATG"
2. "AGGAGA TACCCTT ATG"
I see some expression but the protein runs on a very strange height against the std. ladder. it is supposed to run around 40kDa but it runs around 30-32kDa. The sequencing doesn't really show up any issues.
Do you have any practical tips to improve the productivity? I will try to see if i can get the vector.
Thanks again..
Sometimes proteins run on different position than they should be on SDS-PAGE, it happened to me several times when the expected Mw was off by around 10 kDa, but if you want to be sure you can do peptide mass fingerprinting of your band and as long as you detect peptides from your C-terminus, you should be okay.
Low productivity can have many reasons - starting from low template DNA concetration, ending in the use of rare codons in your mRNA. I use approximately 40 ng/uL final template DNA concentration with home-made cell extract from Rosetta 2 strain (derivative of BL21 (DE3) harbouring plasmid which endodes additional tRNA). If you are still using commercial S30 extract, you can try to make it yourself, it's very easy and cheap to scale up the reaction. For preparation of this extract see Kim et al. J Biotechnol 126 (2006) 554-561),
I hope it helps.
Wow this sounds great.. Thanks for the information.. If i had this information 2 months back it would have been much more easing.. its sooner than never anyways.. Thanks a lot Jiri...I will try to figure out things from the paper..
Cheers
The ideal distance between the RBS and the start codon is 6-8 bases, in your first question it is 16 bases which it is far away from the start codon, in this case it is more likely that gives low level protein expression.
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