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,