[ccp4bb] is codon optimization worth it?
Has anyone encountered a case in which a construct with the native sequence expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding construct with a codon-optimized sequence expressed well? (The gene in question is from cerevesiae) Thanks, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] is codon optimization worth it?
On Fri, 2011-09-30 at 10:49 -0400, Patrick Loll wrote: Has anyone encountered a case in which a construct with the native sequence expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding construct with a codon-optimized sequence expressed well? (The gene in question is from cerevesiae) Wait, isn't Rosetta optimized for mammalian genes, not yeast (but maybe codon bias in yeast matches that)? A sideways suggestion would be to express in yeast. Extra bonus - it smells like beer :) -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] is codon optimization worth it?
Rosetta strains carry a plasmid that supplies several tRNAs that match codons that are rare in E. coli. This is quite different than being explicitly optimized to express mammalian proteins. We (the Center for Eukaryotic Structural Genomics, a PSI-1 and PSI-2 center) used strains with the same tRNA plasmid (either pRARE or pRARE2) to express proteins from yeast, Arabidopsis, some thermophilic eukaryotes, mouse, frog, human and zebrafish proteins. It seemed to work just fine for all of them. On Sep 30, 2011, at 9:55 AM, Ed Pozharski wrote: On Fri, 2011-09-30 at 10:49 -0400, Patrick Loll wrote: Has anyone encountered a case in which a construct with the native sequence expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding construct with a codon-optimized sequence expressed well? (The gene in question is from cerevesiae) Wait, isn't Rosetta optimized for mammalian genes, not yeast (but maybe codon bias in yeast matches that)? A sideways suggestion would be to express in yeast. Extra bonus - it smells like beer :) -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] is codon optimization worth it?
We codon optimised a poorly expressed gene from neisseria meningitides based on a codon usage table derived from the Welch (etal, 2009) paper below. The optimisation is specifically for overexpression in BL21 (DE3). The optimised gene increased protein expression by at least a factor of 10, and changed (somewhat reduced) the degradation pattern we observed. Unfortunately it didn't do anything to improve the folding (ie. we ended up with lots of half-folded, semi-soluble protein). With other neisserial derived proteins we have had an almost undetectable effect. You can't win 'em all. Cheers, Tim Design Parameters to Control Synthetic Gene Expression in Escherichia coli Welch et al, PlosONE 2009 Medizinische Hochschule Hannover Zelluläre Chemie, OE 4330 Zentrum Biochemie Carl-Neubergstr. 1 30625 Hannover -Original Message- From: CCP4 bulletin board on behalf of Patrick Loll Sent: Fri 30.09.2011 16:49 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] is codon optimization worth it? Has anyone encountered a case in which a construct with the native sequence expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding construct with a codon-optimized sequence expressed well? (The gene in question is from cerevesiae) Thanks, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] is codon optimization worth it?
In theory, if the rare codons are all covered by Rosetta's extra tRNAs, codon optimization should not make any difference. In practice it does because frequently it's not codon optimization per se but changing local mRNA structure. - Dima
Re: [ccp4bb] is codon optimization worth it?
To me, the key question would seem to be, if I can't win them all, how many more do I win if I go to the trouble? Brent From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Keys Sent: Friday, September 30, 2011 8:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] is codon optimization worth it? We codon optimised a poorly expressed gene from neisseria meningitides based on a codon usage table derived from the Welch (etal, 2009) paper below. The optimisation is specifically for overexpression in BL21 (DE3). The optimised gene increased protein expression by at least a factor of 10, and changed (somewhat reduced) the degradation pattern we observed. Unfortunately it didn't do anything to improve the folding (ie. we ended up with lots of half-folded, semi-soluble protein). With other neisserial derived proteins we have had an almost undetectable effect. You can't win 'em all. Cheers, Tim Design Parameters to Control Synthetic Gene Expression in Escherichia coli Welch et al, PlosONE 2009 Medizinische Hochschule Hannover Zelluläre Chemie, OE 4330 Zentrum Biochemie Carl-Neubergstr. 1 30625 Hannover -Original Message- From: CCP4 bulletin board on behalf of Patrick Loll Sent: Fri 30.09.2011 16:49 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] is codon optimization worth it? Has anyone encountered a case in which a construct with the native sequence expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding construct with a codon-optimized sequence expressed well? (The gene in question is from cerevesiae) Thanks, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] is codon optimization worth it?
Is there a general consensus that this is true? I’ve heard exactly the opposite, i.e., that codon optimization rarely gives you dramatically improved yields of soluble protein. Are there any published studies on this topic? This seems like something that might come out of one of the SG centers. Brent From: Anastassis Perrakis [mailto:abba...@gmail.com] On Behalf Of Anastassis Perrakis Sent: Friday, September 30, 2011 10:08 AM To: Segelke, Brent W. Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] is codon optimization worth it? Well, codon optimization is not really trouble, it's money. The money are worth it usually anyway, since the optimized genes are easy to clone if you make many constructs out of one gene, as you better do anyway ... Compared with downstream expenses, optimized genes are these days almost always worth the trouble... A. Sent from my iPad On 30 Sep 2011, at 19:02, Segelke, Brent W. segel...@llnl.govmailto:segel...@llnl.gov wrote: To me, the key question would seem to be, if I can’t win them all, how many more do I win if I go to the trouble? Brent From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Keys Sent: Friday, September 30, 2011 8:29 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] is codon optimization worth it? We codon optimised a poorly expressed gene from neisseria meningitides based on a codon usage table derived from the Welch (etal, 2009) paper below. The optimisation is specifically for overexpression in BL21 (DE3). The optimised gene increased protein expression by at least a factor of 10, and changed (somewhat reduced) the degradation pattern we observed. Unfortunately it didn't do anything to improve the folding (ie. we ended up with lots of half-folded, semi-soluble protein). With other neisserial derived proteins we have had an almost undetectable effect. You can't win 'em all. Cheers, Tim Design Parameters to Control Synthetic Gene Expression in Escherichia coli Welch et al, PlosONE 2009 Medizinische Hochschule Hannover Zelluläre Chemie, OE 4330 Zentrum Biochemie Carl-Neubergstr. 1 30625 Hannover -Original Message- From: CCP4 bulletin board on behalf of Patrick Loll Sent: Fri 30.09.2011 16:49 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] is codon optimization worth it? Has anyone encountered a case in which a construct with the native sequence expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding construct with a codon-optimized sequence expressed well? (The gene in question is from cerevesiae) Thanks, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edumailto:pat.l...@drexelmed.edu
Re: [ccp4bb] is codon optimization worth it?
Well, codon optimization is not really trouble, it's money. The money are worth it usually anyway, since the optimized genes are easy to clone if you make many constructs out of one gene, as you better do anyway ... Compared with downstream expenses, optimized genes are these days almost always worth the trouble... A. Sent from my iPad On 30 Sep 2011, at 19:02, Segelke, Brent W. segel...@llnl.gov wrote: To me, the key question would seem to be, if I can’t win them all, how many more do I win if I go to the trouble? Brent From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Keys Sent: Friday, September 30, 2011 8:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] is codon optimization worth it? We codon optimised a poorly expressed gene from neisseria meningitides based on a codon usage table derived from the Welch (etal, 2009) paper below. The optimisation is specifically for overexpression in BL21 (DE3). The optimised gene increased protein expression by at least a factor of 10, and changed (somewhat reduced) the degradation pattern we observed. Unfortunately it didn't do anything to improve the folding (ie. we ended up with lots of half-folded, semi-soluble protein). With other neisserial derived proteins we have had an almost undetectable effect. You can't win 'em all. Cheers, Tim Design Parameters to Control Synthetic Gene Expression in Escherichia coli Welch et al, PlosONE 2009 Medizinische Hochschule Hannover Zelluläre Chemie, OE 4330 Zentrum Biochemie Carl-Neubergstr. 1 30625 Hannover -Original Message- From: CCP4 bulletin board on behalf of Patrick Loll Sent: Fri 30.09.2011 16:49 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] is codon optimization worth it? Has anyone encountered a case in which a construct with the native sequence expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding construct with a codon-optimized sequence expressed well? (The gene in question is from cerevesiae) Thanks, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] is codon optimization worth it?
We recently published something really old-related about it: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0021035 My two cents. Best, Alfredo. Alfredo Torres-Larios, PhD Assistant Professor Instituto de Fisiologia Celular, UNAM Mexico
Re: [ccp4bb] is codon optimization worth it?
Have a look at this paper: http://www.ncbi.nlm.nih.gov/pubmed?term=18289875 the bottom line appears to be that, in most cases, co-transforming with plasmids expressing rare tRNAs is just as good as codon optimizing the gene for your protein of interest. In addition to the financial aspects already touched upon by Tassos, we even had cases where codon optimizing a gene actually completely abolished its expression, presumably by causing havoc at the mRNA level. This obviously won't happen if you co-express rare codon tRNAs... HTH, Luca Luca Jovine, Ph.D. Assistant Professor EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition Center for Biosciences Hälsovägen 7, SE-141 57 Huddinge, Sweden Voice: +46.(0)8.524-81136 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org On Sep 30, 2011, at 19:13 , Segelke, Brent W. wrote: Is there a general consensus that this is true? I’ve heard exactly the opposite, i.e., that codon optimization rarely gives you dramatically improved yields of soluble protein. Are there any published studies on this topic? This seems like something that might come out of one of the SG centers. Brent From: Anastassis Perrakis [mailto:abba...@gmail.com] On Behalf Of Anastassis Perrakis Sent: Friday, September 30, 2011 10:08 AM To: Segelke, Brent W. Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] is codon optimization worth it? Well, codon optimization is not really trouble, it's money. The money are worth it usually anyway, since the optimized genes are easy to clone if you make many constructs out of one gene, as you better do anyway ... Compared with downstream expenses, optimized genes are these days almost always worth the trouble... A. Sent from my iPad On 30 Sep 2011, at 19:02, Segelke, Brent W. segel...@llnl.gov wrote: To me, the key question would seem to be, if I can’t win them all, how many more do I win if I go to the trouble? Brent From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Keys Sent: Friday, September 30, 2011 8:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] is codon optimization worth it? We codon optimised a poorly expressed gene from neisseria meningitides based on a codon usage table derived from the Welch (etal, 2009) paper below. The optimisation is specifically for overexpression in BL21 (DE3). The optimised gene increased protein expression by at least a factor of 10, and changed (somewhat reduced) the degradation pattern we observed. Unfortunately it didn't do anything to improve the folding (ie. we ended up with lots of half-folded, semi-soluble protein). With other neisserial derived proteins we have had an almost undetectable effect. You can't win 'em all. Cheers, Tim Design Parameters to Control Synthetic Gene Expression in Escherichia coli Welch et al, PlosONE 2009 Medizinische Hochschule Hannover Zelluläre Chemie, OE 4330 Zentrum Biochemie Carl-Neubergstr. 1 30625 Hannover -Original Message- From: CCP4 bulletin board on behalf of Patrick Loll Sent: Fri 30.09.2011 16:49 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] is codon optimization worth it? Has anyone encountered a case in which a construct with the native sequence expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding construct with a codon-optimized sequence expressed well? (The gene in question is from cerevesiae) Thanks, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
