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Production of GST tagged fusion proteins in BL21 Star cells

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#1 RVCNick

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Posted 19 February 2013 - 07:07 AM

I'm trying to produce GST fusion proteins to use in pull down experiments. The genes are successfully cloned into pGEX-6P-1 plasmids and confirmed by sequencing and transformed into BL21 Star E.coli for expression. I cannot however seem to produce protein by induction with IPTG. I have tried a range of IPTG concentration up to 2mM and induced at 28oC which was recommended to me. When I Western Blot this using anti-GST antibody I do get bands, 1 at ~26 KDa which is roughly he siz that would be expected for the GST on it's own (there shouldn't be any) which increases in intensity with time but not IPTG concentration. There is a strong band at ~50 KDa which I have no idea what it could be and sometimes I get quite a faint band above this of ~60KDa. The proteins I want to tag are between 54 and 60 KDa so it's probably not these.

I was hoping someone might have had similar problems or may have any general advise for me to produce the proteins that I want.

Thanks

Nick

#2 jerryshelly1

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Posted 19 February 2013 - 07:51 AM

Does any relevant literature possible suggest degradation of your product through protein modifications. It sound like your GST tag is getting cleaved in your cell, that is why you are getting a large signal at your GST expected size. What happens if you use a Ab to look at the endogenous levels of your protein. Do you always see one distinct band or do you see multiple bands?

#3 Pangea

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Posted 13 March 2013 - 12:18 PM

One question to you construct: Do you have additional amino acids after cleavage of your protein from your gst?





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