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Questions on Baculovirus system

Paul Kitts 71774.1102 at CompuServe.COM
Fri Jan 13 04:35:24 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.
If not you can amplify the ORF by PCR or introduce a site by mutagenesis.
 
> 3-  I am planning to use polyhedrin promoter based vectors. The 
> kits that the companies sell are very expensive (almost $500 for 5 
> transfections). Which one is the best choice? 

My opinion on that is strongly biased. :-)

> Is occ based selection too difficult?

Yes, until you have trained your eye to spot the difference.

> Thanks for the help! 

You're welcome.
-= Paul =- 

[These are my opinions, and not necessarily CLONTECH's]
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i Paul Kitts                   Email: paulki at clontech.com  i
i CLONTECH Laboratories Inc.   Phone: 415 424 8222 x415    i
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