[ccp4bb] Off topic: Trouble Shooting on Expression of Membrane protein in Saccharomyces Cerevisiae
I was expressing Membrane protein (100 KD plant cell wall protein with N-terminal His tag) in Saccharomyces Cerevisiae. The vector i am using is PDDGFP modified vector which have URA selection and GAL1 as promoter. I used URA media and Galactose (2%) for induction. I have expressed this construct several times but now for unknown reason it is not expressing. Using exactly the same protocol and reagent. What i already tried are1. Autoclaved as well as filtered media2. Autoclaved as well as filtered Galactose solution3. Fresh transformed cell line from -80C stock4. Newly transformed cell as well.5. Checked the Sequence again and was fine. Please suggest me. M. Obayed Ullah, PhD E.mail: obayed.o...@yahoo.com obayed.o...@gmail.com To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Off topic; Regarding Membrane Protein expression in yeast
Dear, I am planning to express plant membrane protein in Saccharomyces heterogeneous expression system. My question is there any strains which can express any plant membrane protein? For example, if i want to express cellulose synthase and chitin synthases then do i need to use to different strains for these two types of protein or one strain is enough for expressing both the protein? M. Obayed Ullah, PhD E.mail: obayed.o...@yahoo.com obayed.o...@gmail.com
[ccp4bb]
http://www.acondicionamientos-sa.com/fevnzv.php?jrn=uoroea
Re: [ccp4bb] off topic: Another protein forming gel
Hi Tim Thanks for your mail. Both are true. I have not got crystals yet. I have tired different truncated constructs as well as concentration for crystallization. Even i set up the tray at 2 mg/ml but still proteins are not mixing well with the drop solutions rather sitting on the middle of the drop. I dont know what to do with that one. Please suggest me. Cheers M. Obayed Ullah E.mail: obayed.o...@yahoo.com obayed.o...@gmail.com - Original Message - From: Tim Gruene t...@shelx.uni-ac.gwdg.de To: Obayed Ullah obayed.o...@yahoo.com Cc: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, 21 September 2011 6:28 PM Subject: Re: [ccp4bb] off topic: Another protein forming gel -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Obayed, it is not clear to me whether with 'nothing is working' you mean to concentrate the solution even further or whether you mean that you do not get crystals from 7mg/ml. If you refer to the former, I would proceed with crystallisation trials at 7mg/ml. Tim On 09/21/2011 05:57 AM, Obayed Ullah wrote: Hi all I am working with a human protein (not membrane protein) which is forming gel at higher concentration. I am not able to concentrate the protein even more than 7 mg/ml. It become like jelly which is hard to pipette out. I have gone through all the suggestions which you guys proposed very recently for another member. what i mean that i already have tried different temperature, pH, salt concentration, reducing agents (DTT, TCEF, in different concentration), stability analysis. But unfortunately nothing is working. One think i want to try to add some detergent to it. If you guys have any experience about it then please suggest the name and/or kind of detergent for the protein. Any other suggestion also appreciated which might help. cheers M. Obayed Ullah E.mail: obayed.o...@yahoo.com obayed.o...@gmail.com - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFOeaBKUxlJ7aRr7hoRAngZAKD42xoMTzC80OvrOQUwfQ48g4cuRgCg2t5X VVOf9Obqj5eMK5QEwsWbMtQ= =sjqK -END PGP SIGNATURE-
[ccp4bb] off topic: Another protein forming gel
Hi all I am working with a human protein (not membrane protein) which is forming gel at higher concentration. I am not able to concentrate the protein even more than 7 mg/ml. It become like jelly which is hard to pipette out. I have gone through all the suggestions which you guys proposed very recently for another member. what i mean that i already have tried different temperature, pH, salt concentration, reducing agents (DTT, TCEF, in different concentration), stability analysis. But unfortunately nothing is working. One think i want to try to add some detergent to it. If you guys have any experience about it then please suggest the name and/or kind of detergent for the protein. Any other suggestion also appreciated which might help. cheers M. Obayed Ullah E.mail: obayed.o...@yahoo.com obayed.o...@gmail.com
[ccp4bb] off topic: Another protein forming gel
Hi all I am working with a human protein (not membrane protein) which is forming gel at higher concentration. I am not able to concentrate the protein even more than 7 mg/ml. It become like jelly which is hard to pipette out. I have gone through all the suggestions which you guys proposed very recently for another member. what i mean that i already have tried different temperature, pH, salt concentration, reducing agents (DTT, TCEF, in different concentration), stability analysis. But unfortunately nothing is working. One think i want to try to add some detergent to it. If you guys have any experience about it then please suggest the name and/or kind of detergent for the protein. Any other suggestion also appreciated which might help. cheers M. Obayed Ullah E.mail: obayed.o...@yahoo.com obayed.o...@gmail.com
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
[ccp4bb] How to delete loops from Protein for crystallization
Hi all I am thinking to delete flexible loop for crystallization of my protein. But i am not sure how to decide which area i should delete. This protein have around 20% sequence identity with other solved structure. Can anybody suggest me how to proceed for that. Is there any established strategy for that one? Obayed Ullah
