in-vitro post translation modification system

[Dkav]

So...we have an in-vitro translation system with all the things needed to transcribe than translate protein, they also have ones that generate PTM's, but they don't necesarrily generate all the PTM's and are expensive as hell for the scale in which i need the protein. The aim for me is to assess mutated proteins in a cell free system, because growing and maintaining cells is a huge pain in the..big toe. Do you think its feasible to create my own in-vitro PTM system by simply adding all the kinases, phosphatases, organelles that participate in the process? Thanks.

[relaxin]

If you know which kinase is involved in the post translational modification of your protein, certainly you can phosphorylate of protein after cell-free translation. Other modifications may be difficult to do.

[Dkav]

My PI is telling me the same thing, and i don't want to question him lol...excuse my novice here but why do you think its difficult?..Here are the PTM's I need: sulfation, phosphorylation, and a bunch of N-linked glycosylation sites. I guess the challenge would be keeping the integrity of the organelles with their membrane attached enzymes, correct??

[relaxin]

I guess the challenge would be keeping the integrity of the organelles with their membrane attached enzymes, correct??

Yes, some of the post-translational modifications are done inside intact organelles. Once you homogenize the cells, these structures are broken and their normal functions are lost. Cytoplasmic enzymes such as kinases should work in vitro.