Dear Sutapa, I fully agree with Grant, the first question is whether the naturally-produced protein is soluble and whether your protein is not a membrane protein, or a domain, cut out of a much larger protein? The other question is whether your protein is toxic for E.coli and only the bacteria producing it in inclusion bodies survive.
Another thing is to consider is to produce the protein in the same class of organism as the natural producer, e.g. prokaryotic proteins in a bacterial system, mammalian in proteins in mammalian cells etc. My 2 cents, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Vipul Panchal Gesendet: Montag, 3. April 2017 09:06 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility There is also lack of information here. Do you really expect this protein in soluble fraction? Is it a membrane associated or transmembrane protein? Well, I don't have any experience in protein expression using codon optimization. However, Considering the fact that in E.coli it is getting expressed in insoluble fraction, I would recommend to co-express this protein with groEL/ES system. I have plasmid expressing groEL/ES operon. On Mon, Apr 3, 2017 at 12:22 PM, Hansman, Grant <g.hans...@dkfz-heidelberg.de<mailto:g.hans...@dkfz-heidelberg.de>> wrote: Hi, Why don't you try adding GST/MBP tags first? This is a easy quick test. We have a nice fusion (MBP) vector for e.coli expression if you want. Grant From: Sutapa Chakrabarti <chakr...@zedat.fu-berlin.de<mailto:chakr...@zedat.fu-berlin.de><mailto:chakr...@zedat.fu-berlin.de<mailto:chakr...@zedat.fu-berlin.de>>> Reply-To: Sutapa Chakrabarti <chakr...@zedat.fu-berlin.de<mailto:chakr...@zedat.fu-berlin.de><mailto:chakr...@zedat.fu-berlin.de<mailto:chakr...@zedat.fu-berlin.de>>> Date: Monday 3 April 2017 08:49 To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK><mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>>" <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK><mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>>> Subject: [ccp4bb] Using a codon-optimised gene to improve protein solubility Dear All, We're trying to express and purify a 1000 residue long protein and have run into the problem that it is completely insoluble when expressed in E.coli and is not expressed at all in insect cells. The usual tricks for improving solubility in E.coli, such as addition of GST/MBP tags, optimising expression media and induction conditions and use of different cell strains, have not led to any improvement. We are now looking into ordering a codon-optimised synthetic gene for this protein and are trying to decide whether it would be worthwhile to codon-optimise for expression in E.coli (given that the protein was expressed but not soluble) or if we should attempt baculovirus expression again with a gene that has been codon-optimised for insect cells. My question is: has anyone observed an improvement in the solubility of their target protein using a codon optimised gene? I know of several instances where the use of a codon-optimised gene has led to expression where the native gene sequence did not but am unable to find any references for improvement in solubility. Since codon optimisation significantly alters the translation rate of a gene, I believe this should affect solubility as well; but I'd like to know what the community thinks/has observed before I order an exorbitantly priced gene! Thank you in advance, Sutapa -- Sutapa Chakrabarti, Ph.D. Institute of Chemistry and Biochemistry Freie Universität Berlin Takustr. 6 14195 Berlin Germany Phone: +49-(0)30-83875094 -- Vipul Panchal Senior Research Fellow, Respiratory disease and biology, CSIR-IGIB (M)-9540113372
