Amplifying large gene - (Mar/30/2008 )
Hi all,
I am interested in amplifying a large gene ie. 12Kb, sequence of which is avaialbe online on NCBI. This one is annotated as multi-domain enzyme around 240kd. And we are interested in purifying this protein and doing further experiment with this. Though I have little experience in cloning and stuff, I don’t know it would be possible to clone this much big fragment of gene ?...wht should I do, should I design the long primers covering the entire sequence and do PCR with whole DNA as target?........but will it work?.. I mean what is the best way to approach this problem ?
Or the other way I should try to do solve it by proteomics means ie. Doing 2-D of whole culture filtrate, in which this enzyme secretes (where I can use cutt-off filter of100kd for whole protein isolated), and run the remaining protein in native form. But again I guess it will be uncertain way to look at the problem, and its hit and throw method, I don’t know how many spots will come and my protein will be one of them or not?
I am really stuck here with this huge sequence and any help; any suggestion will count for me, as this time, so pls help me out here!
P.S.: - there is no Ab, or enzyme assay is available for this protein.
piearly...... 
I am interested in amplifying a large gene ie. 12Kb, sequence of which is avaialbe online on NCBI. This one is annotated as multi-domain enzyme around 240kd. And we are interested in purifying this protein and doing further experiment with this. Though I have little experience in cloning and stuff, I don’t know it would be possible to clone this much big fragment of gene ?...wht should I do, should I design the long primers covering the entire sequence and do PCR with whole DNA as target?........but will it work?.. I mean what is the best way to approach this problem ?
Or the other way I should try to do solve it by proteomics means ie. Doing 2-D of whole culture filtrate, in which this enzyme secretes (where I can use cutt-off filter of100kd for whole protein isolated), and run the remaining protein in native form. But again I guess it will be uncertain way to look at the problem, and its hit and throw method, I don’t know how many spots will come and my protein will be one of them or not?
I am really stuck here with this huge sequence and any help; any suggestion will count for me, as this time, so pls help me out here!
P.S.: - there is no Ab, or enzyme assay is available for this protein.
piearly......
your gene may be 12 kb, but a 240 kDa protein should only be about 6.5 kb. You're probably looking at the whole gene sequence rather than the mRNA. There are many different ways that you can express your protein and purify it without going to the trouble of doing a 2D gel. Your big questions are whether you want to express your protein in bacteria or a cell line, whether you are concerned that it should retain enzymatic activity, whether you want to visualize the protein, etc. I suggest that you write up a list of the exact experiments that you want to do, do some research on which is the best method to do these experiments and then come back with questions.
Best regards,
smu
I am interested in amplifying a large gene ie. 12Kb, sequence of which is avaialbe online on NCBI. This one is annotated as multi-domain enzyme around 240kd. And we are interested in purifying this protein and doing further experiment with this. Though I have little experience in cloning and stuff, I don’t know it would be possible to clone this much big fragment of gene ?...wht should I do, should I design the long primers covering the entire sequence and do PCR with whole DNA as target?........but will it work?.. I mean what is the best way to approach this problem ?
Or the other way I should try to do solve it by proteomics means ie. Doing 2-D of whole culture filtrate, in which this enzyme secretes (where I can use cutt-off filter of100kd for whole protein isolated), and run the remaining protein in native form. But again I guess it will be uncertain way to look at the problem, and its hit and throw method, I don’t know how many spots will come and my protein will be one of them or not?
I am really stuck here with this huge sequence and any help; any suggestion will count for me, as this time, so pls help me out here!
P.S.: - there is no Ab, or enzyme assay is available for this protein.
piearly......
Hi Piearly,
I suggest you try to look for natural RE sites in your gene, design the flanking primers and clone it in part by part. That way, cloning would be much easier. If your gene is too long, it will break when you are purifying it after PCR and even after digestion.
Hope this helps!