What could be causing degradation of my recombinant enzyme?

01 January 2015 6 5K Report

Hi everyone, thanks for your time.

Even after trying all protease inhibitors, and many separation techniques, I could not remove some contaminating peptides as seen by SDSPAGE. N-terminal sequencing revealed they were in fact degraded or cleaved fragments of my active protein. Theoretical size of active enzyme is 44.7kDa, and fragments are 30 kDa and smaller. I searched cleavage sites using peptide cutter from ExPASy, and no protease cleaves the sights (3 different fragments). They all matched Formic acid degradation, because of an Aspartic acid residue always came before the cleavage site. I don't let my enzyme near formic acid of course and always keep it in a cold, buffered, stable solution. What could be causing this?

Details of production and purification below:

I am producing a 44.7kDa (theoretical) bacterial enzyme, in E.coli BL21(DE3) using pET28a plasmid. Expressing to the periplasm using the native signal sequence (enzyme sequence originally from k.pneumoniae). with a 6x C-term histag.

Induction at 0.6OD with 0.2mM IPTG, 10hours at 30 degrees celcius. (total 500ml of cell culture in LB broth with 50ug/ml Kanamycin, in a 2L shake flask)

Purification all done on ice or 4 degrees Celsius.

cell extraction using bead shock in standard affinity chromatography binding buffer.

Following techniques I tried for purification

affinity chromatography - HistrapHP 5ml column

cation exchange chrom- SPFF 1ml

Hydrophobic interaction chrom. - PhenylHP1ml

Size exclusion chrom. - HiLoad 60/600 75pg 120ml

As my enzyme forms a homodimer in vitro, it seems the degraded fragments are binding to active enzyme, because the 120ml size exclusion column should have separated them.

Thanks!

Steingrimur Stefansson

Hi Kevin, this is an interesting case. If your protein is expressed by bacteria then it should have a N-formyl-Met as the amino terminus.

Could it be that your procedure is releasing the formate from the amino terminus and this is causing the cleavage?

If so, then using buffers containing primary amines might help.

Kevin Sherman

Thank you everyone for your time and help.

Paul - Just estimating by eye from the SDS-PAGE, my enzyme is about 90% pure compared to the degraded fragments -it was enough purity to grow crystals for x-ray analysis, but I would like to try and make it better. I will definitely try your suggestion, as I was considering if this was a good idea or not.

Zdeno - I did not consider this before, I will try it!

Steingrimur - This is very intriguing, and it never would have crossed my mind. I will read about this possibility now!

Again, thank you, its humbling to get such nice responses.

Brigitte Gontero

I was wondering if the pH of the periplasm where you sent your protein (that should be less than 7, periplasm is acid) could be responsible for the cleavage you observed (labile bond of DP is well known, see Nucleic Acid Research 1985, 13 (4) p 11 51 , but there are lots of MS describing this lability). In that case another expression system coudl be tried to express elsewhere than in periplasm

Priyankar Sen

Have you checked exactly at which stage the degradation is taking place? If it is during the purification steps...you should check each step. Anyways you have 90% pure protein, you have protein crystalized. You have not mentioned exactly what drives to do it (crystallization is considered as a purification step).