Re: [ccp4bb] How to express a 95KD FAD protein
try codon optimisation use Rosetta 2 strains fuse to MBP I expressed a yeast protein of ~100 KDa, expression was low until I fused it to MBp (resulting protein ~ 150 KDa)
Re: [ccp4bb] How to express a 95KD FAD protein
With the disclaimer that I have yet to work with a flavoprotein, is there enough FAD around in your culture to allow for proper folding of your protein ? Maybe adding riboflavin to you media, or trying ultimately to refold you protein from inclusion bodies (which will be very pure) in the presence of FAD may work. A quick search in the journal Protein Expression and Purification suggests the latter has been done a few times. Stephen Weeks 2009/6/29 Yong, Wei wy...@mcw.edu Dear all, I know that there are a lot of experts having experience in expressing a big protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding protein (Human protein). I tried very hard but have a bad luck over the past 1.5 years. I list what I did briefly so far. I am looking forward to getting suggestions from you. Thanks a ton in advance. E.Coli 1. Express full-length in pET28a (N-6xhis) No expression 2. Express full-length in pET21b (no-tag) Inclusion body 3. N and C terminus truncated construct Inclusion body (tagged and non-tagged) 4. Co-expression with GroEL/S Inclusion body 5. Co-expression with with cofactors Inclusion body 6. In pMAL vector Not sure Yeast: (No tag) 7. In pPICZb vector in Pichia No expression 8. In GHB30 vector in Saccharomyces cerevisiae No expression I also tried to use different ways to break cells, use detergent, express at low temperature (down to 15 degree), modify IPTG concentration and so on. I do not know what else I can try. Please give me suggestions. Thank you very much. Best wishes Wei Yong
Re: [ccp4bb] How to express a 95KD FAD protein
May be this could help, *Protein Expression and Purification 45 (2006) 191–199* * In vitro activation, purification, and characterization of Escherichia coli expressed aryl-alcohol oxidase, a unique H2O2-producing enzyme. Francisco J. Ruiz-Dueñas, Patricia Ferreira, María J. Martínez, Angel T. Martínez * an in-vitro refolding of a fungal 566 AA FAD-containing protein with FAD added as refolding additive. We have managed to solve the structure. Hope it helps. -- PhD. Israel Sanchez Fernandez EM-lab Departamento de Ciencia de Proteinas CIB-CSIC Madrid España
Re: [ccp4bb] How to express a 95KD FAD protein
Hi, I would like to add one thing to the already good list of suggestions. You can try to express your protein in Lactococcus lactis. We've earlier had successful expression in lactis where it was none or only inclusion bodies in E. coli. See for example: Kunji et al. Biochim Biophys Acta (2003) 97-108, Geertsma and Poolman. Nat Methods (2007) 705-7, Kunji et al. Curr Opin Biotechnol (2005) 546-51. Cheers, Ronnie Berntsson On Jun 29, 2009, at 23:54, Yong, Wei wrote: Dear all, I know that there are a lot of experts having experience in expressing a big protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding protein (Human protein). I tried very hard but have a bad luck over the past 1.5 years. I list what I did briefly so far. I am looking forward to getting suggestions from you. Thanks a ton in advance. E.Coli 1. Express full-length in pET28a (N-6xhis) No expression 2. Express full-length in pET21b (no- tag)Inclusion body 3. N and C terminus truncated construct Inclusion body (tagged and non-tagged) 4. Co-expression with GroEL/ S Inclusion body 5. Co-expression with with cofactors Inclusion body 6. In pMAL vector Not sure Yeast: (No tag) 7. In pPICZb vector in Pichia No expression 8. In GHB30 vector in Saccharomyces cerevisiae No expression I also tried to use different ways to break cells, use detergent, express at low temperature (down to 15 degree), modify IPTG concentration and so on. I do not know what else I can try. Please give me suggestions. Thank you very much. Best wishes Wei Yong
Re: [ccp4bb] How to express a 95KD FAD protein
Hi Wei Yong, Sounds like a tricky situation. A couple of things come to mind: 1) Have you tried expression from a synthetic gene? Sometimes the mRNA is unstable and improving mRNA stability through optimization (synthetic gene) helps. 2) Are you able to look at either human isoforms or orthologs from other species? 3) Have you tried expressing with an N-terminal SUMO tag? I have seen expression with an N-terminal SUMO tag in some cases where expression was previously undetected. 4) Have you tried cell-free expression? 5) You might also consider insect cell expression or expression from mammalian cells 6) Do you get enough protein in some cases that you might try to attempt some refolding from inclusion bodies? Just a couple of avenues to think along. Good luck! Raji On Jun 29, 2009, at 5:54 PM, Yong, Wei wrote: Dear all, I know that there are a lot of experts having experience in expressing a big protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding protein (Human protein). I tried very hard but have a bad luck over the past 1.5 years. I list what I did briefly so far. I am looking forward to getting suggestions from you. Thanks a ton in advance. E.Coli 1. Express full-length in pET28a (N-6xhis) No expression 2. Express full-length in pET21b (no- tag)Inclusion body 3. N and C terminus truncated construct Inclusion body (tagged and non-tagged) 4. Co-expression with GroEL/ S Inclusion body 5. Co-expression with with cofactors Inclusion body 6. In pMAL vector Not sure Yeast: (No tag) 7. In pPICZb vector in Pichia No expression 8. In GHB30 vector in Saccharomyces cerevisiae No expression I also tried to use different ways to break cells, use detergent, express at low temperature (down to 15 degree), modify IPTG concentration and so on. I do not know what else I can try. Please give me suggestions. Thank you very much. Best wishes Wei Yong
Re: [ccp4bb] How to express a 95KD FAD protein
a couple of quick things. This sounds like more of a protein-folding issue. The lack of expression is probably caused by the cells clearing of aggregated protein. You want to have conditions where the protein can properly fold. 1) have you considered periplasmic expression by adding a periplasmic targetting sequence to the N-terminal? It is generally thought, perhaps anecdotally, that the periplasm is better equipped to handle proteins likely to misfold (and presumably cause inclusion bodies). 2) Perhaps a heat shock of your cells prior to induction. Incubate the cells briefly at 42 degrees. This will upregulate the cell's expression of chaperones and other heat shock proteins which may help avoid the inclusion body. 3) Are there any cysteines which might be forming non-specific disulfides? Perhaps this can be resolved by mutagenesis. 4) Last resort. have you tried a snap refold of the inclusion bodies? Dissolve the pellet in urea. Dialyse it away and see if any amount of the protein has refolded spontaneously. good luck Simon Yong, Wei wrote: Dear all, I know that there are a lot of experts having experience in expressing a big protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding protein (Human protein). I tried very hard but have a bad luck over the past 1.5 years. I list what I did briefly so far. I am looking forward to getting suggestions from you. Thanks a ton in advance. E.Coli 1. Express full-length in pET28a (N-6xhis) No expression 2. Express full-length in pET21b (no-tag) Inclusion body 3. N and C terminus truncated construct Inclusion body (tagged and non-tagged) 4. Co-expression with GroEL/S Inclusion body 5. Co-expression with with cofactors Inclusion body 6. In pMAL vector Not sure Yeast: (No tag) 7. In pPICZb vector in Pichia No expression 8. In GHB30 vector in Saccharomyces cerevisiae No expression I also tried to use different ways to break cells, use detergent, express at low temperature (down to 15 degree), modify IPTG concentration and so on. I do not know what else I can try. Please give me suggestions. Thank you very much. Best wishes Wei Yong
Re: [ccp4bb] How to express a 95KD FAD protein
In addition to other excellent suggestions voiced here (insect cells, refolding, etc.) you can attempt expression in constituitive mode - using a mild always-on promoter. Provided that your protein is not too toxic to the cells this may result in gradual accumulation of folded material rather than sudden accumulation of misfolded junk. Artem Nothing is built on stone; all is built on sand, but we must build as if the sand were stone Jorge Luis Borges -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Yong, Wei Sent: Monday, June 29, 2009 4:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to express a 95KD FAD protein Dear all, I know that there are a lot of experts having experience in expressing a big protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding protein (Human protein). I tried very hard but have a bad luck over the past 1.5 years. I list what I did briefly so far. I am looking forward to getting suggestions from you. Thanks a ton in advance. E.Coli 1. Express full-length in pET28a (N-6xhis) No expression 2. Express full-length in pET21b (no-tag) Inclusion body 3. N and C terminus truncated construct Inclusion body (tagged and non-tagged) 4. Co-expression with GroEL/S Inclusion body 5. Co-expression with with cofactors Inclusion body 6. In pMAL vector Not sure Yeast: (No tag) 7. In pPICZb vector in Pichia No expression 8. In GHB30 vector in Saccharomyces cerevisiae No expression I also tried to use different ways to break cells, use detergent, express at low temperature (down to 15 degree), modify IPTG concentration and so on. I do not know what else I can try. Please give me suggestions. Thank you very much. Best wishes Wei Yong
