I have some problem with my his-tagged recombinant protein expression.
My goal is to create antibodies against canine MMP-9. I have successfully extracted the coding region of a section of the molecule which should have good antigenic properties.
Ligation with pet24a(+) vector was successfull
Transformation into top10 E.coli and then BL21 E.coli was successfull
SO far so so good, but here is my two problems :
In both small scale and large scale expression, i have 2 proteins that show up.
My protein should be 21.8 Kda and i get two big marks, one at 22kDa and one at 25kDa
In the "no induction" sample, taken before Iptg induction, the 25kDa is absent but the 22kDa is very slightly present (leaky expression?), and then at 3h, 6h, 9h, 12h and 20h post-induction, both are present with the 25kDa being slightly bigger.
I did a western blot using anti-his-tag Ab, i get a strong signal at 22kDa and a very weak but present signal at 25kDa.
Also, may or may not be related to this, both protein are impossible to purify using sepharose beads. 95% of it goes in the flow-through and does not bind to beads (the beads are functionnal as they worked with another protein)
I'd like to get some insight and opinions on my problem,
thank you for your help,
here is more additional information :
vector : pet24(a) with kanamycine resistance gene
insert : around 500bp
theorical size of protein including his-tag : 21.8kDa
Medium : LB broth + kanamycine
Expression protocol :
refresh an overnight culture of bacteria at 1/20 ration in 200ml medium, incubate at 37°C 250rpm until OD is between 0,6 and 1 then induce with 1mM IPTG. Harvest 6h post induction for large scale.
The protein is at 99% insoluble, so i extract it with a binding buffer containing 6M Urea. In such conditions, the protein should not be able to fold on itself and hide its his-tag....(i think)
Please excuse my mistakes, english is not my first language