Hey, So i have a 39kDa Membrane Protein, cloned into pRSET C and transformed into C43, sequenced to makes sure everything is there and made a glycerol stock of.

From that glycerol stock i start a culture overnight, next day i pellet it, resuspend in fresh LB and inoculate a culture and let it grow to OD 0.6-0.7 where i add 1mM IPTG and let it grow for 4 more hours for induced and without iptg for uninduced. all at 37C. All standard stuff.

I tried lysis in about 500ul of 2 different buffers for 4ml cell cultures:

Buffer A: 25 mM TRIS-Cl, 2 mM EDTA, pH 7.6. 5mg/ml lysozyme

Buffer B: 40mM Na2HPO4, 300mM NaCl, 10mM Imidazole. ph 8. 1mg/ml lysozyme

Incubated at RT for about an hour( checked cells under phase microscope and i did see a bit of lysis but most of the cells were still intact with both buffers even tho the lysis was more with the EDTA but i observed the E coli aggregating into huge clusters in that buffer)

So lysozyme alone isn't working for lysis for me, EDTA or no EDTA

*Worth saying cell suspension turbidity was obervable higher for uninduced samples, i'll bring this up again later.

Sonicated both solution until turbidity was gone.

Centrifuged at 20000g for 20min

Separated into supernatant and pellet

Supernatant stored at 4C till next day, Pellet incubated in 8M Urea in respective buffers overnight.

Next day i run 12% SDS-Page and ive attached a picture. my protein is about 39KDa

Lane M , Molecular markers, Lane 1 supernatant uninduced Buffer B, Lane 2 supernatant induced Buffer B, Lane 3 supernatant uninduced Buffer A, Lane 4 supernatant induced Buffer A, Lane 5 pellet uninduced Buffer B, Lane 6 pellet induced Buffer B, Lane 7 Pellet uninduced Buffer A, Lane 8 pellet induced Buffer A

Could it be the basal expression is toxic? and like i mentioned uninduced cells also grew much more turbid. Could it be plasmid containing cells are dying upon induction?

I will try 1% glucose before induction to see if that will resolve the issue or at least give me a difference between induced and uninduced. Also will run an empty plasmid transformed cell.

Will Also try to Ni purify the pellet , in case the arrow ive indicated might be my protein. but i doubt it.

Is it possible they aren't toxic and just aren't being expressed, or solubilized by 8M Urea?

Western is not an option if you will recommend it.

PS Should i just go for a cell free system such as

PURExpress supplemented with liposomes?