Re: [ccp4bb] Protein expression (codon bias)
I did crystallise a protein expressed in M. smegmatis a while ago (early 90's)! The cloning was done by Ying Zhang:https://www.jhsph.edu/faculty/directory/profile/786/ying-zhangIt might be worth dropping him a line. That's all I can suggest, sorry!!Good luck.Jon CooperOn 7 Jul 2020 10:07, Matthew Snee wrote: Hi all I'm designing genes for _expression_ in M. smegmatis (safe host for Mtb proteins), but its not possible (or advisable due to the GC content) to optimise for mycobacterial _expression_. Would anyone with experience be able to tell me if its fine to stick with the E. coli codon optimisation, or if there is an advantage going with b. subtilis (or another G+ organism that is in Thermo's list). Best wishes Matthew. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
Re: [ccp4bb] protein expression in human cells
Hi all, If anybody is interested in non-viral stable expression, we have a piggybac-based, doxycycline-inducible system. It is the reference 40 in the lentiviral paper that Tomas directed to. We’d be happy to distribute the plasmids. Zhijie > On Jan 25, 2020, at 3:26 AM, Tomas Malinauskas > wrote: > > Hi Gloria, > > two key papers describing expression (transient and lentivirus-based) > of proteins in HEK293 cells we use to make milligrams of proteins for > crystallization and cryo-EM: > > Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1243-50. Epub > 2006 Sep 19. > A time- and cost-efficient system for high-level protein production in > mammalian cells. > Aricescu AR, Lu W, Jones EY. > PMID: 17001101 > > Nat Protoc. 2018 Dec;13(12):2991-3017. doi: 10.1038/s41596-018-0075-9. > Lentiviral transduction of mammalian cells for fast, scalable and > high-level production of soluble and membrane proteins. > Elegheert J, Behiels E, Bishop B, Scott S, Woolley RE, Griffiths SC, > Byrne EFX, Chang VT, Stuart DI, Jones EY, Siebold C, Aricescu AR. > PMID: 30455477 > > Hope that helps, > Tomas > > > Dr. Tomas Malinauskas > University of Oxford > Wellcome Centre for Human Genetics > Division of Structural Biology > Roosevelt Drive > Oxford OX3 7BN > United Kingdom > to...@strubi.ox.ac.uk > tomas.malinaus...@gmail.com > >> On Fri, Jan 24, 2020 at 11:50 PM Gloria Borgstahl >> wrote: >> >> Hello CCP4-ers, >> I was wondering what people have found to be the best human cell line >> expression system for making a large quantity of purified recombinant >> protein. >> Any information and protocols would be greatly appreciated. >> Happy 2020, Gloria >> >> >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] protein expression in human cells
Hi Gloria, two key papers describing expression (transient and lentivirus-based) of proteins in HEK293 cells we use to make milligrams of proteins for crystallization and cryo-EM: Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1243-50. Epub 2006 Sep 19. A time- and cost-efficient system for high-level protein production in mammalian cells. Aricescu AR, Lu W, Jones EY. PMID: 17001101 Nat Protoc. 2018 Dec;13(12):2991-3017. doi: 10.1038/s41596-018-0075-9. Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins. Elegheert J, Behiels E, Bishop B, Scott S, Woolley RE, Griffiths SC, Byrne EFX, Chang VT, Stuart DI, Jones EY, Siebold C, Aricescu AR. PMID: 30455477 Hope that helps, Tomas Dr. Tomas Malinauskas University of Oxford Wellcome Centre for Human Genetics Division of Structural Biology Roosevelt Drive Oxford OX3 7BN United Kingdom to...@strubi.ox.ac.uk tomas.malinaus...@gmail.com On Fri, Jan 24, 2020 at 11:50 PM Gloria Borgstahl wrote: > > Hello CCP4-ers, > I was wondering what people have found to be the best human cell line > expression system for making a large quantity of purified recombinant protein. > Any information and protocols would be greatly appreciated. > Happy 2020, Gloria > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] protein expression in human cells
For general information, HEKs were derived from a legally aborted foetus the in the Netherlands in 1973. Not "killed children". (As a side note, such use of emotive language is counter-productive and often the tool of those who seek remove reproductive rights and autonomy from women. I'm certain we wouldn't want to be tarred with that brush.) That said, use CHOs if you have an issue with HEKs. The only potential problem would be if what you wanted to study required authentic human post-translational modifications, and even then, I imagine CHOs would still be a suitable surrogate in the majority of cases. Cheers, Dave -- Dr David C. Briggs Senior Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK == about.me/david_briggs From: Petr Kolenko Sent: Saturday, January 25, 2020 7:10:06 AM To: David Briggs ; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] protein expression in human cells Dear colleagues, I wonder if there were a bit less controversial possibility. No matter if that was less efficient. Would there be an option of using human cell lines that do not origin from killed children? As far as I know, the HEK cells do. Sometimes, the science can be pretty cruel. I am sorry for opening of this topic on a crystallographic forum, but so far, nobody has convinced me that I am allowed to work with these cell lines from a humanity (and Christian) point of view. Best regards, Petr From: CCP4 bulletin board On Behalf Of David Briggs Sent: Saturday, January 25, 2020 7:49 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein expression in human cells Hi Gloria, Another vote here for HEK 293 Expi or Freestyle. The off-the-shelf transfection reagents are super expensive, but I make my own (happy to share protocols with anyone interested) and we normally get a few mgs of protein per litre of suspension culture -- certainly enough for crystallography. If budget is an issue, semi-stable transfection of adherent HEK293s is much cheaper but the turnaround from vector to protein is much slower (a few weeks rather than a few days), and growing up panels of mutants can be quite tedious. Cheers & good luck Dave -- Dr David C. Briggs Senior Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK == about.me/david_briggs From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Rezaul Karim <1e364c8f16de-dmarc-requ...@jiscmail.ac.uk<mailto:1e364c8f16de-dmarc-requ...@jiscmail.ac.uk>> Sent: Saturday, January 25, 2020 1:31:02 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: Re: [ccp4bb] protein expression in human cells In our lab, we see Expi293F works very good. Thanks, Reza Md. Rezaul Karim Graduate student, PhD Program in Integrated Biomedical Sciences Morsani College of Medicine University of South Florida E-mail: reza...@yahoo.com<mailto:reza...@yahoo.com>, rez...@health.usf.edu<mailto:rez...@health.usf.edu> Phone: +1-954-937-8487 On Friday, January 24, 2020, 4:51:11 PM EST, Gloria Borgstahl mailto:gborgst...@gmail.com>> wrote: Hello CCP4-ers, I was wondering what people have found to be the best human cell line expression system for making a large quantity of purified recombinant protein. Any information and protocols would be greatly appreciated. Happy 2020, Gloria To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1=02%7C01%7C%7C8d9cb69fc73147febb4308d7a1659dc1%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637155330121696788=qqIBXCSemuXhA57kmQV0LSsi951HzMK%2BcdBE0Pr3YvI%3D=0> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1=02%7C01%7C%7C8d9cb69fc73147febb4308d7a1659dc1%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637155330121706793=RKNGSjCxiqW039sHw7FZu8rGyh%2FXmWYUYd8%2BkBkgb4w%3D=0> The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2
Re: [ccp4bb] protein expression in human cells
Dear colleagues, I wonder if there were a bit less controversial possibility. No matter if that was less efficient. Would there be an option of using human cell lines that do not origin from killed children? As far as I know, the HEK cells do. Sometimes, the science can be pretty cruel. I am sorry for opening of this topic on a crystallographic forum, but so far, nobody has convinced me that I am allowed to work with these cell lines from a humanity (and Christian) point of view. Best regards, Petr From: CCP4 bulletin board On Behalf Of David Briggs Sent: Saturday, January 25, 2020 7:49 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein expression in human cells Hi Gloria, Another vote here for HEK 293 Expi or Freestyle. The off-the-shelf transfection reagents are super expensive, but I make my own (happy to share protocols with anyone interested) and we normally get a few mgs of protein per litre of suspension culture -- certainly enough for crystallography. If budget is an issue, semi-stable transfection of adherent HEK293s is much cheaper but the turnaround from vector to protein is much slower (a few weeks rather than a few days), and growing up panels of mutants can be quite tedious. Cheers & good luck Dave -- Dr David C. Briggs Senior Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK == about.me/david_briggs From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Rezaul Karim <1e364c8f16de-dmarc-requ...@jiscmail.ac.uk<mailto:1e364c8f16de-dmarc-requ...@jiscmail.ac.uk>> Sent: Saturday, January 25, 2020 1:31:02 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: Re: [ccp4bb] protein expression in human cells In our lab, we see Expi293F works very good. Thanks, Reza Md. Rezaul Karim Graduate student, PhD Program in Integrated Biomedical Sciences Morsani College of Medicine University of South Florida E-mail: reza...@yahoo.com<mailto:reza...@yahoo.com>, rez...@health.usf.edu<mailto:rez...@health.usf.edu> Phone: +1-954-937-8487 On Friday, January 24, 2020, 4:51:11 PM EST, Gloria Borgstahl mailto:gborgst...@gmail.com>> wrote: Hello CCP4-ers, I was wondering what people have found to be the best human cell line expression system for making a large quantity of purified recombinant protein. Any information and protocols would be greatly appreciated. Happy 2020, Gloria To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1=02%7C01%7C%7Cdd4cd6f62475425a3afa08d7a1364e92%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637155126940120399=XEh8QKGrpCzQSR7EFXkx9uwNaJQGeUEZz6dmh%2FUQgso%3D=0> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1=02%7C01%7C%7Cdd4cd6f62475425a3afa08d7a1364e92%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637155126940120399=XEh8QKGrpCzQSR7EFXkx9uwNaJQGeUEZz6dmh%2FUQgso%3D=0> The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] protein expression in human cells
Hi Gloria, Another vote here for HEK 293 Expi or Freestyle. The off-the-shelf transfection reagents are super expensive, but I make my own (happy to share protocols with anyone interested) and we normally get a few mgs of protein per litre of suspension culture -- certainly enough for crystallography. If budget is an issue, semi-stable transfection of adherent HEK293s is much cheaper but the turnaround from vector to protein is much slower (a few weeks rather than a few days), and growing up panels of mutants can be quite tedious. Cheers & good luck Dave -- Dr David C. Briggs Senior Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK == about.me/david_briggs From: CCP4 bulletin board on behalf of Rezaul Karim <1e364c8f16de-dmarc-requ...@jiscmail.ac.uk> Sent: Saturday, January 25, 2020 1:31:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein expression in human cells In our lab, we see Expi293F works very good. Thanks, Reza Md. Rezaul Karim Graduate student, PhD Program in Integrated Biomedical Sciences Morsani College of Medicine University of South Florida E-mail: reza...@yahoo.com, rez...@health.usf.edu Phone: +1-954-937-8487 On Friday, January 24, 2020, 4:51:11 PM EST, Gloria Borgstahl wrote: Hello CCP4-ers, I was wondering what people have found to be the best human cell line expression system for making a large quantity of purified recombinant protein. Any information and protocols would be greatly appreciated. Happy 2020, Gloria To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1=02%7C01%7C%7Cdd4cd6f62475425a3afa08d7a1364e92%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637155126940120399=XEh8QKGrpCzQSR7EFXkx9uwNaJQGeUEZz6dmh%2FUQgso%3D=0> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1=02%7C01%7C%7Cdd4cd6f62475425a3afa08d7a1364e92%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C0%7C637155126940120399=XEh8QKGrpCzQSR7EFXkx9uwNaJQGeUEZz6dmh%2FUQgso%3D=0> The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Protein expression
Hi, I’ve received a number of concurring suggestions. Some have requested more detail about the experiments. Here are the details. 1. Cells: BL21(DE3) 2. Plasmid: pET28a (T7 promoter) 3. Media: TB 4. Protein: cytoplasmic 5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce with 1mM IPTG, grow 16hrs at 20degC, centrifuge. 6. Lysis method: Sonication via micro-tip/macro-tip. Small culture produces soluble protein where as large culture produces insoluble protein. We first thought it may be due to poor lysis, thus equivalent amount of cells from 3 and 500ml cultures were lysed with the micro-tip. Same results were observed. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York 160 Convent Ave, MR-1135 New York, NY 10031 (212) 650-6070 rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu On Mar 19, 2015, at 11:15 PM, John Fisher johncfishe...@gmail.commailto:johncfishe...@gmail.com wrote: Hi Reza. Clearly nobody needs to know anything about what protein you are specifically working on; that being said, in order to avoid a potentially endless email string of expert advices, please include everything detail-wise regarding your expression system, culture conditions, induction, and lysis method for BOTH the 3 ml and 500 ml expression trials. You will get, I imagine, amazing advices likely specific enough to solve the problem without your having to chase your tail. Best, John Fisher John C. Fisher, M.D./PhD On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu wrote: Hi, We can express quite a bit of soluble protein when growing 3ml cultures. However, the protein becomes insoluble (inclusion bodies) when we scale up to 500ml cultures. Has anyone experienced such a problem, and found a solution to it? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070tel:212-650-6070
Re: [ccp4bb] Protein expression
Hi Reza, A few month ago I had a the exact same problem and we checked everything we could think of but without any improvement. Finally we were able to solve the problem only by subcloning the ORF into another plasmid (pET9a or pET29b). The big difference being the His tag position (C-ter or N-ter) might be critical for some protein solubility in large scale protein expression. Hope it could help Abbas Le 20/03/2015 11:56, Reza Khayat a écrit : Hi, I’ve received a number of concurring suggestions. Some have requested more detail about the experiments. Here are the details. 1. Cells: BL21(DE3) 2. Plasmid: pET28a (T7 promoter) 3. Media: TB 4. Protein: cytoplasmic 5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce with 1mM IPTG, grow 16hrs at 20degC, centrifuge. 6. Lysis method: Sonication via micro-tip/macro-tip. Small culture produces soluble protein where as large culture produces insoluble protein. We first thought it may be due to poor lysis, thus equivalent amount of cells from 3 and 500ml cultures were lysed with the micro-tip. Same results were observed. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York 160 Convent Ave, MR-1135 New York, NY 10031 (212) 650-6070 rkha...@ccny.cuny.edu mailto:rkha...@ccny.cuny.edu On Mar 19, 2015, at 11:15 PM, John Fisher johncfishe...@gmail.com mailto:johncfishe...@gmail.com wrote: Hi Reza. Clearly nobody needs to know anything about what protein you are specifically working on; that being said, in order to avoid a potentially endless email string of expert advices, please include everything detail-wise regarding your expression system, culture conditions, induction, and lysis method for BOTH the 3 ml and 500 ml expression trials. You will get, I imagine, amazing advices likely specific enough to solve the problem without your having to chase your tail. Best, John Fisher John C. Fisher, M.D./PhD On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat rkha...@ccny.cuny.edu mailto:rkha...@ccny.cuny.edu wrote: Hi, We can express quite a bit of soluble protein when growing 3ml cultures. However, the protein becomes insoluble (inclusion bodies) when we scale up to 500ml cultures. Has anyone experienced such a problem, and found a solution to it? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070 tel:212-650-6070 -- El Sahili Abbas Ph.D. Student (+33) 01.69.82.42.49 abbas.el-sah...@lebs.cnrs-gif.fr Solange Moréra Team Structural Microbiology Enzymology Institut for Integrative Biology of the Cell (I2BC) Biochemistry, Biophysic and Structural Biology (B3S) Departement CNRS-Gif campus Bat 34, Avenue de la Terrasse 91190 Gif sur Yvette
Re: [ccp4bb] Protein expression
Hi, Cultures are being properly cooled prior to induction (water bath supplemented with ice). Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070 From: lieh low [mailto:liehy...@gmail.com] Sent: Friday, March 20, 2015 8:06 AM To: Reza Khayat Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein expression Reza, someone might have mentioned this, it takes longer time to cool down larger culture, we turn down the temp of the shaker when OD is about 0.4. For some protocol, we even use ice to cool the flask down before induction. You might also want to consider a lower induction temp, like 16degC. Maybe your protein is just temp sensitive. ray On Fri, Mar 20, 2015 at 6:56 AM, Reza Khayat rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu wrote: Hi, I’ve received a number of concurring suggestions. Some have requested more detail about the experiments. Here are the details. 1. Cells: BL21(DE3) 2. Plasmid: pET28a (T7 promoter) 3. Media: TB 4. Protein: cytoplasmic 5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce with 1mM IPTG, grow 16hrs at 20degC, centrifuge. 6. Lysis method: Sonication via micro-tip/macro-tip. Small culture produces soluble protein where as large culture produces insoluble protein. We first thought it may be due to poor lysis, thus equivalent amount of cells from 3 and 500ml cultures were lysed with the micro-tip. Same results were observed. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York 160 Convent Ave, MR-1135 New York, NY 10031 (212) 650-6070tel:%28212%29%20650-6070 rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu On Mar 19, 2015, at 11:15 PM, John Fisher johncfishe...@gmail.commailto:johncfishe...@gmail.com wrote: Hi Reza. Clearly nobody needs to know anything about what protein you are specifically working on; that being said, in order to avoid a potentially endless email string of expert advices, please include everything detail-wise regarding your expression system, culture conditions, induction, and lysis method for BOTH the 3 ml and 500 ml expression trials. You will get, I imagine, amazing advices likely specific enough to solve the problem without your having to chase your tail. Best, John Fisher John C. Fisher, M.D./PhD On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu wrote: Hi, We can express quite a bit of soluble protein when growing 3ml cultures. However, the protein becomes insoluble (inclusion bodies) when we scale up to 500ml cultures. Has anyone experienced such a problem, and found a solution to it? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070tel:212-650-6070
Re: [ccp4bb] Protein expression
Reza, someone might have mentioned this, it takes longer time to cool down larger culture, we turn down the temp of the shaker when OD is about 0.4. For some protocol, we even use ice to cool the flask down before induction. You might also want to consider a lower induction temp, like 16degC. Maybe your protein is just temp sensitive. ray On Fri, Mar 20, 2015 at 6:56 AM, Reza Khayat rkha...@ccny.cuny.edu wrote: Hi, I’ve received a number of concurring suggestions. Some have requested more detail about the experiments. Here are the details. 1. Cells: BL21(DE3) 2. Plasmid: pET28a (T7 promoter) 3. Media: TB 4. Protein: cytoplasmic 5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce with 1mM IPTG, grow 16hrs at 20degC, centrifuge. 6. Lysis method: Sonication via micro-tip/macro-tip. Small culture produces soluble protein where as large culture produces insoluble protein. We first thought it may be due to poor lysis, thus equivalent amount of cells from 3 and 500ml cultures were lysed with the micro-tip. Same results were observed. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York 160 Convent Ave, MR-1135 New York, NY 10031 (212) 650-6070 rkha...@ccny.cuny.edu On Mar 19, 2015, at 11:15 PM, John Fisher johncfishe...@gmail.com wrote: Hi Reza. Clearly nobody needs to know anything about what protein you are specifically working on; that being said, in order to avoid a potentially endless email string of expert advices, please include everything detail-wise regarding your expression system, culture conditions, induction, and lysis method for BOTH the 3 ml and 500 ml expression trials. You will get, I imagine, amazing advices likely specific enough to solve the problem without your having to chase your tail. Best, John Fisher John C. Fisher, M.D./PhD On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat rkha...@ccny.cuny.edu wrote: Hi, We can express quite a bit of soluble protein when growing 3ml cultures. However, the protein becomes insoluble (inclusion bodies) when we scale up to 500ml cultures. Has anyone experienced such a problem, and found a solution to it? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070
Re: [ccp4bb] Protein expression
Hi Reza, In addition to the many useful suggestions already made, I would suggest lowering the final concentrations of IPTG. In many cases, 1mM IPTG interferes with expression levels and/or solubility. This suggestion does not address your concern for why things become ugly in going from 3mL to 500mL (for which there can be many many explanations), but its worth a try before you head off to make many more clones. Cheers and best wishes, Raji On Thu, Mar 19, 2015 at 3:33 PM, Reza Khayat rkha...@ccny.cuny.edu wrote: Hi, We can express quite a bit of soluble protein when growing 3ml cultures. However, the protein becomes insoluble (inclusion bodies) when we scale up to 500ml cultures. Has anyone experienced such a problem, and found a solution to it? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070
Re: [ccp4bb] Protein expression
Dear Reza, It sounds to me like an aeration issue. I don't of course know how the 3 ml culture is grown, but if say the small culture is less perfectly aerated and slightly anaerobic, slower growth would mean slower metabolism and slower protein production as well so things do not build up so easily in inclusion bodies. I've had something like this happen in the past. The case I'm talking about was that the protein did not express in aerobic conditions (4 L flask, 500 mL of culture), when I increased the liquid volume to 2-3L in the flask, the culture became slightly anaerobic and protein production turned on. Perhaps this is your case as well. Might be worth a shot! Cheers, -Tiit -- Tiit Lukk Staff Scientist CHESS 200L Wilson Lab Ithaca, NY 14853 phone: 607-255-5717 e-mail: tl...@cornell.edu url: http://www.chess.cornell.edu On Thu, Mar 19, 2015 at 3:33 PM, Reza Khayat rkha...@ccny.cuny.edu wrote: Hi, We can express quite a bit of soluble protein when growing 3ml cultures. However, the protein becomes insoluble (inclusion bodies) when we scale up to 500ml cultures. Has anyone experienced such a problem, and found a solution to it? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070
Re: [ccp4bb] Protein expression
Question: you're taking a 3-ml equivalent out of 500 ml culture, and processing it as if it was a 3ml culture? Or are you basing the result on processing the entire 500ml? Reason I ask this is simply to make sure your extraction/purification timing is the same in both cases. It matters! Assuming that the protein really is not soluble at 500ml and is somewhat soluble at 3ml - which expression set up are you using? IPTG or autoinduction? more details is better. For example, if you use IPTG - how do you determine when to induce - OD? or some other measure? The advice to change aeration is solid. Try it both ways, meaning up or down - it could go either way depending on the difference in your set up. A 3ml culture in a 24-well block at 800 rpm is MUCH better aerated than 500ml culture in non-baffled 1L flask at 250rpm. You also may want to play with temperature, and if you're not using AIM - try it out. Artem - Cosmic Cats approve of this message On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat rkha...@ccny.cuny.edu wrote: Hi, We can express quite a bit of soluble protein when growing 3ml cultures. However, the protein becomes insoluble (inclusion bodies) when we scale up to 500ml cultures. Has anyone experienced such a problem, and found a solution to it? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070
Re: [ccp4bb] Protein expression
Hi Reza. Clearly nobody needs to know anything about what protein you are specifically working on; that being said, in order to avoid a potentially endless email string of expert advices, please include everything detail-wise regarding your expression system, culture conditions, induction, and lysis method for BOTH the 3 ml and 500 ml expression trials. You will get, I imagine, amazing advices likely specific enough to solve the problem without your having to chase your tail. Best, John Fisher John C. Fisher, M.D./PhD On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat rkha...@ccny.cuny.edu wrote: Hi, We can express quite a bit of soluble protein when growing 3ml cultures. However, the protein becomes insoluble (inclusion bodies) when we scale up to 500ml cultures. Has anyone experienced such a problem, and found a solution to it? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070
Re: [ccp4bb] Protein expression, purification and crystallization
If you are eligible, I'd highly recommend the EMBO courses in Europe or the CSHL courses in the US on those subjects. Reading is a good start but will not be enough if you want to do real work.for strategy considerations, there is always Ch 3 and 4 in le livre. Best, BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hena Dutta Sent: Thursday, July 21, 2011 10:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein expression, purification and crystallization Dear Members of CCP4BB and PHENIXBB, Can you suggest a reliable website where I can get basic and advanced information on protein expression, purification and crystallization. I like to read on the monitor. Thanking you in advance... Hena
Re: [ccp4bb] protein expression
Hi Chen, In this case, it seems that linker region is of great importance for the proper folding of the two linked domains. I have not much experience in linker region design, generally use (GS)5-10 times. However it depends on individual case. Anyone who has successful experience in linker region design might share your success with others. Thanks a lot. Regards, Donghui On 3/5/08, Daniel Jin [EMAIL PROTECTED] wrote: Hi, I have a protein with two independently folded domains. I can express either one in bacteria with pretty good expression yield. However, when I put them together with a linker, the expression drops significantly. I can barely see any soluble protein and most of it is now inclusion bodies. I used the same bacteria strain and expression/purification conditions. Is it normal? Any suggestion about how to improve? Many thanks. Best, Chen -- Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.http://us.rd.yahoo.com/evt=51733/*http://mobile.yahoo.com/;_ylt=Ahu06i62sR8HDtDypao8Wcj9tAcJ
Re: [ccp4bb] protein expression problem
I have been trying to express a rat protein in bacteria. The MBP-fusion expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag only gave inclusion bodies. The problem is that all protein runs in the void volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl), no matter it is the intact MBP-fusion or cleaved sample. There is no Cys on this protein so there is unlikely any disulfide bond related problem. Anything I can do before I throw away this construct and try insect or mammalian cells? Thanks. First of all, using a carrying protein (like GST, MBP) can be disconcerting. These proteins are very soluble and can solubilize an insoluble protein in testing condition. So you have something soluble but your protein of interest can be misfolded or can precipitate when the carrying protein was cleaved. So keep in mind that a soluble carried protein is not always a good protein. After this consideration you have a wide range of conditions to test. - Solubility of your MBP-fusion: ultracentrifugation, thermal shift assay (in different pH, salt, salt concentration), micro-dialysis - Folding of your MBP-fusion: circular dichroism, 1D NMR (if you can, compare with MBP alone) - Aggregation/monodispersity of your MBP-fusion: DLS (in different pH, salt, salt concentration) - For gel filtration assays don't forget that MBP can dimerize The second part of your tests can be expression conditions. Sometimes low but native expression is better than high but carried or insoluble expression. - Medium - Temperature - Host cells (different E. coli, yeast, insect cells...) - Inducing strength - Co-expression with ligands or chaperones And the last but not the least part of your tests can be refolding. Inclusion body expression is the first step of your purification. If you have his-tagged protein in inclusion body a one step purification can be performed in denaturing conditions. A wide range of refolding conditions can be tested: - Flash dilution - Dialysis - Refolding by slow gradient on an Ni column (if you have an his tag) - pH, salt, detergent conditions - Chaperones OD at 340 nm can monitor the refolding efficiency. Good luck Michel
Re: [ccp4bb] protein expression problem
I wholly agree with the below. I am not sure how well E.coli can correctly fold snaky misfolded/unfolded protein that are chaperoned by folded tags! Not to rule out that tags do it sometimes... Folded by association for insoluble proteins has often not worked well for me. Sometimes, when it 'works' for me and my colleagues, removal of the tag leads to insoluble protein/aggregation etc. I am dealing with SUMO tagged proteins that have enhanced solubility but severe degradation issues. Screening for pH, buffers and all the good-old stuff folks have suggested here is a good approach. Sometimes autoinduction protocols, which keep from 'overexpression' of protein in the cell might be an approach to explore after the above tests. Good luck! Raji First of all, using a carrying protein (like GST, MBP) can be disconcerting. These proteins are very soluble and can solubilize an insoluble protein in testing condition. So you have something soluble but your protein of interest can be misfolded or can precipitate when the carrying protein was cleaved. So keep in mind that a soluble carried protein is not always a good protein.
Re: [ccp4bb] protein expression problem
Chen and David, Before adding detergent, be forewarned that the MPB in many fusions will not bind to an amylose column in the presence of most detergents, particularly maltoside detergents. It has been the bane to us so we have engineered MBP vectors with His tags to deal with this. What you might try, as suggested, the NDSBs or the addition of glycylglycine (to make 0.5-1.0M) to the growth media just before innoculation (don't worry about sterility with good antibiotic selection; autoclaving will just make a brown mess). The glycylglycine trick can reduce aggregation. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: [EMAIL PROTECTED] On Jan 22, 2008, at 2:23 AM, David Briggs wrote: Hi Chen, You could try adding some detergent or other solubilising agent (eg NDSBs) to your buffer. Have you tried other pHs? If you are sat near to or on the pI of your protein, it will be at its least soluble and more likely to aggregate. I've had protein behave like yours at pH 7.5 but behave perfectly (i.e. monodisperse) at pH 5.5. As you can get you protein in inclusion bodies, have you considered doing an inclusion body prep (using 'bugbuster' or something similar) and then trying some refolding protocols? Jungbauer A, Kaar W. Current status of technical protein refolding.J Biotechnol. 2007 Feb 20;128(3):587-96. Some people have had success with SUMO tags as well. HTH, Cheers, David On 22/01/2008, Daniel Jin [EMAIL PROTECTED] wrote: Hi, I have been trying to express a rat protein in bacteria. The MBP- fusion expressed at very high level (~ 40 mg/L) while the GST- fusion and His-tag only gave inclusion bodies. The problem is that all protein runs in the void volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl), no matter it is the intact MBP-fusion or cleaved sample. There is no Cys on this protein so there is unlikely any disulfide bond related problem. Anything I can do before I throw away this construct and try insect or mammalian cells? Thanks. Best, Chen Never miss a thing. Make Yahoo your homepage. -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile
Re: [ccp4bb] protein expression problem
Hi Chen, Since you recognize that this is a protein expression problem, the best way to get return of your investment of efforts is to get soluble expression instead of trying to solubilize the protein down stream. Many good suggestions have been proposed. I just want to add one more thing for you to try. Lower the induction temperature to 16C (or any temperature between 10-30C). With all the tricks to make protein soluble in E. coli, low induction temperature is the only universal method that works. Accelagen has made all kinds of proteins and we have systematically tried many variables. Only induction temperature matters for solubility, everything else just gives you more or less proteins. If your promoter is not tightly controlled, you may just let the E. coli grow to saturation without induction, at low temperature of course. It worked well for pGEX based vectors. Many proteins give little soluble expression in E. coli. In those cases, Baculovirus expression system is a great alternative. Once you have your gene in a transfer vector, it takes about 3 weeks to know the soluble expression level in insect cells and another 3 weeks to harvest 10 L cell pellets. Currently technology allows you to scale up to 100s L in an additional couple weeks. The material cost is actually similar to E. coli expression. It's faster than trying to figure out how to solubilize a protein. Good luck. Chun Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 mailto:[EMAIL PROTECTED] [EMAIL PROTECTED] www.accelagen.com _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Daniel Jin Sent: Monday, January 21, 2008 10:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein expression problem Hi, I have been trying to express a rat protein in bacteria. The MBP-fusion expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag only gave inclusion bodies. The problem is that all protein runs in the void volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl), no matter it is the intact MBP-fusion or cleaved sample. There is no Cys on this protein so there is unlikely any disulfide bond related problem. Anything I can do before I throw away this construct and try insect or mammalian cells? Thanks. Best, Chen _ Never miss a thing. Make Yahoo http://us.rd.yahoo.com/evt=51438/*http:/www.yahoo.com/r/hs your homepage.
Re: [ccp4bb] protein expression problem
Hi Chen, You could try adding some detergent or other solubilising agent (eg NDSBs) to your buffer. Have you tried other pHs? If you are sat near to or on the pI of your protein, it will be at its least soluble and more likely to aggregate. I've had protein behave like yours at pH 7.5 but behave perfectly (i.e. monodisperse) at pH 5.5. As you can get you protein in inclusion bodies, have you considered doing an inclusion body prep (using 'bugbuster' or something similar) and then trying some refolding protocols? Jungbauer A, Kaar W. Current status of technical protein refolding.J Biotechnol. 2007 Feb 20;128(3):587-96. Some people have had success with SUMO tags as well. HTH, Cheers, David On 22/01/2008, Daniel Jin [EMAIL PROTECTED] wrote: Hi, I have been trying to express a rat protein in bacteria. The MBP-fusion expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag only gave inclusion bodies. The problem is that all protein runs in the void volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl), no matter it is the intact MBP-fusion or cleaved sample. There is no Cys on this protein so there is unlikely any disulfide bond related problem. Anything I can do before I throw away this construct and try insect or mammalian cells? Thanks. Best, Chen Never miss a thing. Make Yahoo your homepage. -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile
Re: [ccp4bb] Protein expression in Minimal media (M9)
I had a similar situation, i.e. the cells wouldn't grow past an OD of 0.1 on minimal media, but in my case I did get protein expression. The way I got around it was as follows. I grew the cells up in minimal media plus the amino acids that suppress methionine biosynthesis, plus L-methionine. For whatever reason, the cells behaved much better in not quite minimal media. Then, when they were near 0.6 OD, I spun them down and resuspended them in media with Selenomethionine instead of L-Met. It worked for me (i.e. ~100% SeMet incorporation, structure solved, etc.), but I only needed to solve a growth problem, not an expression problem. It might work for you, and you could try a dry run without burning up precious SeMet. _ Eric A. Toth, Ph.D. Assistant Professor Department of Biochemistry and Molecular Biology Marlene and Stewart Greenebaum Cancer Center University of Maryland School of Medicine 108 North Greene St. Baltimore, MD 21201 Email: [EMAIL PROTECTED] Phone: x-410-706-5345 Fax: x-410-706-8297 http://www.umaryland.edu/bmb/faculty/toth.html http://crystal.umaryland.edu -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Wednesday, April 18, 2007 10:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein expression in Minimal media (M9) Hello everybody, Sorry for an offtopic question. I am trying to express a protein in M9 minimal media for Selenomet incorporation. When grown in LB this protein expressed very well and got good crystals. Diffraction was upto 2 A. I am having a hard time expressing the same protein in Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in minimal media and eventually got no protein expression. It looks like the cells are not growing or taking very long to grow. The cell line I am using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use RosettaBlue (DE3). It worked very well in LB, but having a hard time while expresing the same in minimal media using Rosetta Blue. Has anybody tried expression in minimal media using Rosetta Blue cell line? I am planning to try overnight induction. Any suggestions would be greatly appreciated. Thanks, Manish * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. *
Re: [ccp4bb] Protein expression in Minimal media (M9)
Manish, I also had a similar problem getting cells (C41 (DE3) in my case) to grow in minimal media. To get around this problem, I took cells from an agar plate and grew them in a small volume (5 mL) of the minimal media. Once that culture got thick, I then inoculated 200 mL of minimal media with 0.5 mL of the 5 mL culture. I would let this grow, and I used this as my overnight culture to inoculate my large flasks. Long story short, the cells seemed happier adjusting to the minimal media in a smaller volume first. Cheers, Jamie Wallen Hello everybody, Sorry for an offtopic question. I am trying to express a protein in M9 minimal media for Selenomet incorporation. When grown in LB this protein expressed very well and got good crystals. Diffraction was upto 2 A. I am having a hard time expressing the same protein in Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in minimal media and eventually got no protein expression. It looks like the cells are not growing or taking very long to grow. The cell line I am using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use RosettaBlue (DE3). It worked very well in LB, but having a hard time while expresing the same in minimal media using Rosetta Blue. Has anybody tried expression in minimal media using Rosetta Blue cell line? I am planning to try overnight induction. Any suggestions would be greatly appreciated. Thanks, Manish * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. *
Re: [ccp4bb] Protein expression in Minimal media (M9)
Are you adding vitamins to your M9 minimal? RosettaBlue is thiamin requiring and can not grow in the absence of thiamin. The thiamin requirement is so low that you can often get slow growth to a low OD based on residual thiamin in the cells, but you will not get robust growth. Also, minimal recipes vary pretty drastically from one another, your system may prefer one of the other recipes out there. Personally, I have never really liked the standard M9 minimal recipe. Contrary to another poster, I found that my cells tended to grow better in minimal medium if I pre-adapted them to minimal by growing my starters in the same minimal that I intended to use (with Met instead of SeMet). However, I prefer to stick with high dilutions (1:1000-1:100) so that may be the difference. For years I used the MM/CA recipe from Pryor and Leiting, Protein Expression and Purification, 1997 v10 pg 309. This recipe works well and can be adapted for SeMet growth by subbing out the casamino acids for a mixture of amino acids. For the past few years I've been using the autoinduction recipes from Studier, Protein Expression and Purification, v41 pg 207. I have found that these work fantastically well (as long as you don't need to express at really reduced temperature...I only use them down to 20° C). There are recipes for SeMet incorporation in there as well. Best of luck, Cynthia On Apr 18, 2007, at 10:34 PM, [EMAIL PROTECTED] wrote: Hello everybody, Sorry for an offtopic question. I am trying to express a protein in M9 minimal media for Selenomet incorporation. When grown in LB this protein expressed very well and got good crystals. Diffraction was upto 2 A. I am having a hard time expressing the same protein in Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in minimal media and eventually got no protein expression. It looks like the cells are not growing or taking very long to grow. The cell line I am using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use RosettaBlue (DE3). It worked very well in LB, but having a hard time while expresing the same in minimal media using Rosetta Blue. Has anybody tried expression in minimal media using Rosetta Blue cell line? I am planning to try overnight induction. Any suggestions would be greatly appreciated. Thanks, Manish * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. * Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844
Re: [ccp4bb] Protein expression in Minimal media (M9)
Manish, Assuming that you need the minimal medium for Se, have you tried the regular heavy atom derivatives? You already have diffracting crystals... M9 medium can be slightly difficult. However, no one says that you *have* to use minimal medium such as M9 (again, assuming that you're doing Se-Met). There are numbers of defined media compositions out there, many with very good growth characteristics. Finally, you can spike the M9 with a small quantity of yeast extract. This will give your culture a decent initial boost and the amount of normal methionine in the extract should be relatively small so it will all get eaten up while the cells are still in early log phase. Regards, Artem Hello everybody, Sorry for an offtopic question. I am trying to express a protein in M9 minimal media for Selenomet incorporation. When grown in LB this protein expressed very well and got good crystals. Diffraction was upto 2 A. I am having a hard time expressing the same protein in Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in minimal media and eventually got no protein expression. It looks like the cells are not growing or taking very long to grow. The cell line I am using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use RosettaBlue (DE3). It worked very well in LB, but having a hard time while expresing the same in minimal media using Rosetta Blue. Has anybody tried expression in minimal media using Rosetta Blue cell line? I am planning to try overnight induction. Any suggestions would be greatly appreciated. Thanks, Manish * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. *
Re: [ccp4bb] Protein expression in Minimal media (M9)
Manish, In practice, we have found that it is very helpful to grow up an overnight starter culture in minimal media to acclimatize the cells for growth under minimal conditions. (Cells transferred from LB to M9 do not perform well for overexpression). We inoculate 1 L of modified M9 (see below) with 10 mL of an overnight culture (centrigued, washed, and ressuspended in M9) in the same medium. In addition--and even though it should not be required for many strains of E. coli--we have found that he inclusion of thiamin, 5-10 ug/L, greatly enhances the rate of cell growth, but it may still takeup to 8 hr for cells to grow to OD600 = 0.6 for induction. Overexpression overnight (12-18 hr) may be required to get optimal cell density and overexpression yield. Using this protocol we get excellent overexpression yields of protein. Cheers, ___ Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Thursday, April 19, 2007 10:31 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein expression in Minimal media (M9) Manish, Assuming that you need the minimal medium for Se, have you tried the regular heavy atom derivatives? You already have diffracting crystals... M9 medium can be slightly difficult. However, no one says that you *have* to use minimal medium such as M9 (again, assuming that you're doing Se-Met). There are numbers of defined media compositions out there, many with very good growth characteristics. Finally, you can spike the M9 with a small quantity of yeast extract. This will give your culture a decent initial boost and the amount of normal methionine in the extract should be relatively small so it will all get eaten up while the cells are still in early log phase. Regards, Artem Hello everybody, Sorry for an offtopic question. I am trying to express a protein in M9 minimal media for Selenomet incorporation. When grown in LB this protein expressed very well and got good crystals. Diffraction was upto 2 A. I am having a hard time expressing the same protein in Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in minimal media and eventually got no protein expression. It looks like the cells are not growing or taking very long to grow. The cell line I am using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use RosettaBlue (DE3). It worked very well in LB, but having a hard time while expresing the same in minimal media using Rosetta Blue. Has anybody tried expression in minimal media using Rosetta Blue cell line? I am planning to try overnight induction. Any suggestions would be greatly appreciated. Thanks, Manish * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. *
