pls make some of your points clear
1)is the organism the yeast or bacteria from which isolated?
2)do you know its gene sequence? or its upstream or downstream sequence? perhaps no.
3)how r u sure that you isolated the right protein?
4) r u sure that your mass spec was error free?
please respond to these queries which will make others and me comfortable to answer.
Regards
Aditya
Dear Aditya and friends
My organism is yeast from klyvermyces spp
no i don't know the sequence of gene? i purified my killer toxin protein by anion exchnage chromatography, got one peak then did SDS it showed one clear band with killing activity as i checked its activity after elution and sds
i get my protein sequence by mass from company i have no relative sequence how can i make it sure that the sequence send by company has erro while it show killing activity
Thank you for your information Mr. Aslam Muhammad. let me think about it a bit and then I will try my best to suggest you something relevant. And it will be my pleasure if any of my suggestions be of any help to you.
thank you friend i am waiting for you preicious suggestions.
i am thinking that may be my protein is new discovery and i can call it noval killer toxin what do you think?
Aslam
dear Aslam muhammad
I have certain views regarding your experiment related to finding whether the killer protein is coded by gdna or plasmid.
1)there is a high chance that protein is encoded by plasmid because some strains of kluyeromyces contain pGKL1 and pGKL2 plasmid that encodes killer protein. It may be due to certain mutations that you are getting new a different killer protein.
2)you can cure plasmid from the cell either by optimal uv treatment or any other method and see if the protein concenteration is lowered in such strains for few generations. if it does then it may indicate that plasmid contain killer protein.
3)another approach may be that you isolate the plasmid and then insert it into non-encoding strain of competent kluyeromyces and check for protein expression.
competent cells may be prepared by treating the cell by ethylene glycol and dimethyl sulfoxide(DMSO) and rapid freezing(-70 degree). slight warming of the cell(30 to 40 degree) in shaking condition in liquid media may make it competent.
4)size of the plasmid and the protein may be compared with the normal pGKL1 or pGKL2 plasmid and its proteins respectively to get an idea about any mutation taking place.
5)later for the confirmation, the plasmid may be fragmented by different and appropriate type of restriction enzyme and then ligated to appropriate plasmid and cloned into E.coli or other bacteria and check for protein production and thus for final confirmation.
Hope this write up will help you in your experiment. and I will be highly encouraged if any of my suggestion helps you and I find my name in the acknowledgement part of your paper. ha ha ha. By the way best of luck for your research. I will be very happy if i can be of any help to you in future.
I also invite you to join my newly created group (innovations) and please tell your friends about it.
Best Regards
Aditya Parekh.
dear Aditya parekh
thank you very much for detail suggestion
i have purified my killer toxin protein and send it for sequence and after doing blast it doesn't resemble with any other known killer toxin but about 14 % sequence hace lipase similarty.very interestingly it have no lipase axctivity too. then i design degenrate prime form the sequence i obtain by my ourified protein then did pcr and get three band i have cut them i send them for sequence lets se what will be the result and which will be mine?
i have that paper you coded as refrence for plasmid killer toxin. i apprciate your effort for me and science too. i will be intouh regarding my study and keep on distrubing you i hope you never mind it hahahahha
as reagard to acknowledge it would be my pleasure to acknowledge you in my paper but it is not promise as you know i am phD student therefore totaly under superise so it depends upon situation.
regards
Aslam
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