Questions on Baculovirus system

Paul Kitts 71774.1102 at CompuServe.COM
Tue Jan 10 03:20:06 EST 1995


Huseyin Kucuktas <kucukhu at mail.auburn.edu> writes:
> Hello,
> 
> I am trying to express a protein which is supposed to be a product
> of a 5406 bp DNA fragment. However the actual ORF is small and 
> the gene reads on the complementary strand.
> I HAVE SOME QUESTIONS!
> 1-  Maximum how many extra bases I can have before the ATG 
> codon of my "gene"? 

You should avoid having any other ATG, regions with strong secondary
structure, and long stretches of G/C upstream from the initiating 
ATG. Other than these features there is no clear correlation between
length of untranslated region and expression level - make it as 
short as is conveniently possible.

> 2-  It appears that I do not have a lot of desirable restriction 
> sites around the beginning of the ORF, how am I going to get rid 
of  
> the extra bases?

Use whatever site is closest to the ATG. Since your ORF is small you
may even find a 4-base cutter that does not cut within the ORF. If 
you can cut within 50 bp (or even 100 bp) of the ATG, I would try 
that.



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