Fusion proteins
magda umm
Monday, 31 August 2009 19:27 UTC
Hi, I’m not very advanced in the cloning so maybe someone of you could help me to understand the reason of my problem. I want to clone 2 genes of heme proteins, so I added the second gene to the first one, present on the plasmid. I didn’t remove the stop codon of the first gene because i put the ribosome binding site before the second one. In this way I would like to have 2 separated proteins. As heme proteins are transfered to periplasm I don’t need any tag. I checked also the presence of right sequences in the plasmid amplificated by PCR. SDS-PAGE of bacteries growth showed the right size of the proteins which should be red but they are white. Did somebody do something like this?
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Replies
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Anonymous
Hi Magda,
that sounds like you are expressing mammalian proteins in bacteria. Did you check for all sorts of posttranslational modifications that may be necessary for protein function and/or structure and that will not take place in bacteria? And, since you are expressing proteins with bound iron, will iron also be bound in the bacterial environment?
And, did you sequence your construct? I would say that PCR or restriction digestion based quality control is not sufficient, since even point mutations, f.i. from your cloning PCR or primer synthesis, situated for example in the iron binding domain may have a huge impact.
For the color: You are conducting SDS based PAGE, which as a core feature denatures proteins before seperation. During denaturion, the proteins will lose any bound iron and by that also lose their color. Maybe you can make a western blot to check wether you have the right protein in the band?
Hope that helps!
Cheers,
Henning
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