If you are referring to the beta-lactamase enzyme KPC-2, it is a soluble periplasmic protein of a Gram negative bacterium. It can be expressed as a soluble protein in E. coli using a periplasmic secretion signal sequence.

You probably funcatinally inactive protein expressed that is the reason to have proteins in pellets. 

What temperature are expressing your proteins at?

Is is 37C?

Hi Abid

In the past I have expressed recombinant enzyme from plasmid in E Coli strain BL 21 DE3. Protein ending up as an insoluble fraction or inclusion body. This is a common problem caused fundamentally by the fact that you are re engineering the big to express high amounts of a foreign protein under changing temp conditions ( which induces heat shock proteins). Apart from titrating IPTG as you have done 2 other things definitely helped

1. Express the protein at a lower temp for a longer period ; in our case 18C for 24 hrs 

2. Prior to induction at 18c grow the bugs to an exact ID 600 density ( which I will provide in a proper SOP Monday ), add medium plus antibiotics then heat shock by placing culture at 42C for 20 to 30 min then ice. Finally commence the 18 to 24 hr induction

i will send exact details in a protocol Monday but this always increased the proportion of expressed protein in the soluble fraction

First all thaks to all for given me quick and helpfull response

  Dear Adam B Shapiro.

Thanks alot for your answer. yes i am working on beta lactamase gram negative bacteria, can you provide me full detail about E. coli periplasmic secretion signal sequence.

 Dear Laurence Stuart Hall

Thanks Alot for your courageous  help in my experiment. yes i did iptg induction in different lower temp like 16, 18, 20 25 and 30 for overnight incubation but did not find result satisfactory. if possible please send me your protocol which you have used and got result. my email id : [email protected]

I would appreciate if you could the same.

thanks

ABID 

Dear Charandeep Singh.

Thanks alot for your kind help. i have use protien expression iptg induction on different temp like 16,20,25 and 30 c. yes my protien is active form and total AA IS 293.

Thanks 

Abid

Dear Abid

I will send to the email address you have provided

Best wishes

Dear Abid, you have already gotten good advice but let me share my story... We also had difficulties getting our protein to the soluble fraction, and variations of the purification techniques did not solve the problem. What finally worked for us was that during the fermentation, we used a combination of low temperature (+16), low IPTG concentration (0.1 mM), and addition of the cofactor necessary for the correct conformation of the protein.

Good luck for the experiments!

Dear Tarja Kokkola ,

Thanks alots  for your nice answer of my question, really your idea may be solve my puzzle.  previously I did twice my experiment 16c and 0.1 mM IPTG  on over night incubation but  i was not add any cofactor,might be cofactor solve my problem.

Thanks

with regards

ABID ALI

KPC-2 does not have a cofactor. It is a serine beta-lactamase.

Dear Adam B Shapiro,

i am thinking about to change my vector then may be get good result.can you sher me have any idea about periplasmic protien vector or competent cell.

Thanks

abid ali

Here are some general guide lines on keeping recombinant expressed proteins solubilised. As mentioned in my previous answer heat shocking  prior to prolonged expression (24hrs) @ a low temp (16-18C) plus lower concentrations of IPTG and also lower starter densities of transformants prior to 24 hours @ 16-18C can help

In addition find a link to my drop box folder with the above plus more background info on optimising fusion expression

https://www.dropbox.com/sh/8rxn1rj1m9k49qg/AACKlTaAWA2wkVldcaXRpR-ta?dl=0

Dear.Laurence Stuart Hall 

Thanking you very much for your kind email and your effort.

You might try a pelB leader sequence.

https://en.wikipedia.org/wiki/PelB_leader_sequence