I'm a student that do a project by using yeast for recombinant protein production, and I'm out of this field for cell-free protein production, but I have some doubt.
I see many kits from many company that use very short time for 30 minutes or 2 hours for protein production that very convenient. If compare with microorganism that use a longer time for day or many days. And microorganism have to construct the plasmid that have many elements that affect the expression level. Have to choose promoter, terminator, selection marker, culture medium, the induction or cultivation system, optimization for protein in flask culture or bioreactor etc. Therefore, I see many disadvantages for use microorganism for recombinant protein production.
So, for many time that I think about this question. Why In Vitro (Cell-free) recombinant protein synthesis don't use instead of microorganisms in the industrial scale? May be a price? or any reasons please suggest me.
Thank you so much,
Tatpong
My guess would be that it is far easier and less expensive to use living microorganisms than an in vitro translation system. The cells replicate quickly and they can produce the desired protein when grown in a relatively inexpensive medium. To use an in vitro translation system, you have to prepare the S30 extract, so you have to grow and extract cells anyway. Then you have to provide a complex and expensive mixture of nucleotides, amino acids and other things. At the end, you still have to purify the protein out of the translation extract just as you would from the whole cells.
One time when cell-free translation is useful is when you want to make a protein that is toxic to the cells.
My guess would be that it is far easier and less expensive to use living microorganisms than an in vitro translation system. The cells replicate quickly and they can produce the desired protein when grown in a relatively inexpensive medium. To use an in vitro translation system, you have to prepare the S30 extract, so you have to grow and extract cells anyway. Then you have to provide a complex and expensive mixture of nucleotides, amino acids and other things. At the end, you still have to purify the protein out of the translation extract just as you would from the whole cells.
One time when cell-free translation is useful is when you want to make a protein that is toxic to the cells.
Price of making the cell free lysate and quantity of protein produced.
Hi
During intracellular protein expression, after translation process some post translation modifications (such as glycosylation) occur on polypeptide backbone which these modifications are critical for protein function. Intracellular organelles such as endoplasmic reticulum and transgolgi complex overtake those modifications in the cell but cell-free reaction mixture lacks those organelles and as a result its product wont be completely functional.
Yes, a lot of economical and technical barriers limit the industrialization of cell free proteins, as mentioned by the above nice answers.
But I believe the in vitro synthesis of artificial proteins will become technical and economical feasible and will have broader applications - e.g. (1) synthesis of non-natural proteins which not only incorporate amino acids but also other functional structures which cannot be recognized by natural protein production factory; or (2) for those proteins which are not feasible for the microbial systems, as mentioned by Dr. Shapiro.
I can remember how expensive it is to synthesize cell free DNA when I first step into the molecular lab at around 2000 (I guess the situation is worse before PCR technique) . So please have an eye on frontier studies in this field, the breakthrough techniques like protein PCRs are waiting around the corners and they will make cell free artificial proteins feasible to every lab.
Also ease of purification is a factor. Most systems have protein excreted outside the cells into media with minimal protein added. Thus cells perform initial purification of your protein, since most of cell protein stays inside.
With in vitro synthesis all components of the system are in the same soup. Possibly immobilization of components on solid supports could help but it will also affect kinetics.
Cells renew their own resources for protein production using relatively simple components of animal-free media while in vitro production systems deplete themselves without renewal and produce more impurities instead.
Because of an economical issue, it is better to use micro-organism instead of cell free protein production.
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