I am working on beta lactamase. I have constructed a clone containing beta lactamase gene, transformed and expressed protein using Penicillin in E. coli. Now need to isolate protein. I have tried different lysis buffers. but on SDS-PAGE, no band is detected. Can anyone suggest any good method for isolation. SDS-PAGE protocol is already standardised. now Just to get the protein. I checked the concentration also using bradford reagent but showing very minute concentration
Hi there,
I guess your BLA is meant to be periplasmic if using resistance to penicillin as a selection marker. Unless you have cloned the gene without signal peptide.
First, you need to find out whether the beta-lactamase is being expressed, and if so, whether the protein is soluble. Next, you need to lyse the cells. A French Press would be my suggestion, but sonication or bed-beater can also be used. Freeze-thawing the cell pellet and adding lysozyme may also be helpful additions. Ultracentrifuge the resulting extract, and if the lysis was successful and the protein is soluble, it should be in the supernatant. Hopefully, it will show up as a strong band on SDS-PAGE of the supernatant.
I'm wondering whether using penicillin to induce expression is the best approach. If the protein is expressed, it will destroy the penicillin, so induction may be self-limiting. Also, the cells probably have an AmpC beta-lactamase gene, which will also be induced by penicillin, so you could end up expressing 2 beta-lactamases. It might be better to use IPTG induction.
Dear Muneeb K Hamza,
Here I have attached some pdf file and a book on isolation and purification of proteins which will be very help full to you in your work. another pdf file is on isolation or purification of proteins from inclusion bodies , Expression of recombination proteins. You can see all these things. and hope you will short out your problems.
best Regards,
Dr Anand
You might try the method described in the attached publication.
Good luck and regards,
Susan Tzotzos
Dear Adam,
Thanks alot for your answer. Actually we have tried IPTG also as an inducer. Anyway I will try lysozyme. Once again thank you
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques