we have got a bacteria which showed very good protease activity . Now, we want to clone the gene and express the protein in Ecoli, but we dont know the gene how we can clone and express.
Why not run different purification steps with different methods (CEX, AEX, HIC) to maybe purify the activity and then run a protein fingerprint on the active sample to identify putative genes via the protein sequence?
Alternatively, you could construct an expression library and plate it on plates with a protein substrate. There are plenty of recipes to be found on Internet for such plate assays. Recombinants harbouring and expressing protein genes are then easily detected ad picked up for further characterization.
Dr Pierre suggested a nice procedure. Using a shotgun library construction you may be able to identify the recombinants carrying the protease genes. Then you can send the recombinant vector for sequencing.
Another way is to identify your bacteria strain using 16S rRNA, from there you can search for protease genes in a close related strain that have been fully sequenced. Primers can then be designed base on the conserved regions of the identified sequenced of the protease genes.These may not be very accurate.
Another way was as suggested above, purifying the protein and sequencing it, then you can design your primers based on the protein sequence. All of the afore mentioned procedures are cumbersome
I did rather suggest you sequence the whole genome of your new strain (although more expensive but it is more guaranteed). Nowadays you can get the whole genome sequenced and annotated with not more than 4,000 USD.
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