We have been trying to overexpress and purify full length gyrase A from E coli, but we seem to have a knack for getting a cleaved product, even during expression. We've tried a few different clones, but not the "original" Hallett et al plasmids. Would anyone know where we could source JMtacA and JMtacB? Alternatively, if anyone knows a trick for gyrA I'd love to hear it!
Thanks in advance
well, if you want fully activated and folded enzyme you should use advanced expression vector, mammalian cell
Previous publications have been successful with E coli, and it is a bacterial protein. I don't think mammalian cells are the way to go..... thanks for your help though!
Ah Christopher - good point - we've tried a variety of BL21 DE3 strains, but I don't think a protease deficient one. Any suggestions there? Thanks!
Hello! Do you see a cleaved product only or you have some full-length protein? I had some experience with gyrase purification (from E. coli and Pseudomonas strains), I used N-terminal His-tag and pET expression vectors but I've never seen truncated protein. Purification of GyrA should be straightforward and yield is pretty good, purification of GyrB is a little bit more tricky because of relatively low yield. What is the size of cleaved product? Did you sequenece your plasmid?
Hi Mikhail! Yes, it does appear even during expression, but increases to almost total after cell lysis - we've tried a variety of E coli hosts and two different plasmid sets (we have sequenced the ones we are using, but we do not have the JMtac series). We have a His tag on each end, so can track the cleavage into two products (CTD and remaining), although we haven't done further analysis to confirm those are not contaminants (His-tags are not great for blotting). It does seem a straightforward expression in other lab's hands, which is why I'm confused by our results.
Thanks for the help, and we will keep investigating.
Dear Dr,unfortunately I have not sufficient expertis in this field.
The company Inspiralis Ltd specialises in purifying and selling topoisomerases, and E. coli DNA gyrase is one of their specialities. Their technical support team is very helpful as well so you might want to drop them an email with your question (or just buy the final protein!).
www.inspiralis.com
Here is the follow-up
We did fix this problem by finally sequencing the plasmid and realizing that the wrong clone was sent. Rookie mistake, won't happen on my watch again. ;)
Thanks everyone for the thoughts!
(As a side note we are customers of Inspiralis and are making great progress with their products. For structural studies we need larger quantities than we can afford to buy!)
Not uncommon. My experience is ca. 50% of isolates sent from university labs are not correct. Mislabeling, passed around, etc. Learned the hard way.
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