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How to eliminate inherent proteins contamination during protein purification?

protein expression protein purification Ni-NTA purification Rosetta galactosidase

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#1 Ravish Godse

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Posted 15 October 2019 - 03:55 AM

Hello people,


I have two issues:


1. For one recombinant galactosidase gene, we have cloned the gene in pET28a and expressing in E. coli Rosetta(DE3). I can see over-expression on SDS-PAGE but after purification with 150 mM imidazole, many inherent proteins are also getting eluted out due to which we are getting inherent protein activity along with our protein of interest. How can eliminate these inherent protein contamination?


2. For another recombinant galactosidase gene, we cloned the gene in TOPO system and tried to express it in E.coli BL21 Star (DE3) and Rosetta (DE3). We have got the gene sequenced and it matches with our reference sequence but it is not getting expressed in both E.coli BL21 Star (DE3) and Rosetta (DE3) using IPTG. What can be the probable solution for it?


Thanks in advance for your insights,


#2 mdfenko


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Posted 18 October 2019 - 01:53 PM

1. If the sizes of the contaminating proteins are different enough from your protein of interest then you can separate them on size exclusion chromatography. If not, then you can try ion exchange chromatography or chromatofocusing.


you may also be able to separate with density gradient centrifugation.


whichever method you select will depend on the characteristics of the proteins involved. You may have to use more than one to sufficiently purify your protein of interest for your purposes.


as a last resort, you can isolate the protein of interest by electrophoresis, cut the band and elite, inject into mice to produce antibodies, purify and use for immunoprecipitation (including magnetic isolation) or immunoaffinity chromatography.

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