Cell-free expression system - the gospel of difficulty protein expression

In 1964, two people pioneered the expression of in vitro protein. The names of these two people must not be unfamiliar—Matthew and Nilenberg. Because of their innovative thinking, humans have deciphered 64 translational codons encoding amino acids. Since then, in vitro protein expression has begun to be of concern to the scientific community, but at the time the system protein expression was low, short duration, and poor stability, making it unable to further develop.

After Spirin et al. optimized the protein synthesis yield in the mid-1980s, the in vitro protein expression system, the cell-free expression system, began to pay more attention to the bioengineering field and gradually developed various types of cell-free expression systems. There are four commonly used: E. coli extract cell-free expression system, yeast extract cell-free expression system, wheat germ extract cell-free expression system and rabbit reticulocyte extract cell-free expression system.

Use cell extracts (such as E. coli, yeast cells, wheat germ, rabbit red blood cells, etc.) to provide RNA polymerase, tRNA, ribosomes, amino acids, initiation factors, elongation factors, termination factors, etc., and add expression template plasmids or The PCR product continuously supplies the substrate amino acid, energy-ATP and the like, and at the same time removes reaction by-products by dialysis and the like, and performs protein expression in an in vitro environment. This technique is called cell-free expression technology.

Several major advantages of cell-free expression:

1. The biggest advantage of the cell- free expression system is to avoid the influence of the intracellular environment on cell expression, and artificially control the reaction conditions and processes. Jin Kairui's cell-free expression system optimizes the system, such as Tag optimization, M optimization, and K optimization, to obtain optimal expression conditions.

Tag optimization for yua3 protein

M optimization of VACV I5 protein

K optimization of TMEM65 protein

2. Significantly shorten the experimental period. The ordinary expression process takes 2~3 days, and the cell-free expression process is greatly shortened to 1~2 hours, which saves a lot of time cost, and solves the urgent need for those experimental partners who are eager to graduate.

3. It is also easy to solve proteins that are difficult to express in conventional cells, such as membrane proteins and toxic proteins. Nearly one-third of mammalian genomes are composed of membrane proteins, and about half of them can be used as drug targets. The study of these target membrane proteins and the development of potential bio-targeted drugs are of great significance for disease pathogenesis and drug development.

Expression of seven transmembrane proteins ADRB2

Expression of toxic protein ExoU

4. Protein complexes can be prepared to study protein interactions. Proteins expressed by cell-free systems can be used in downstream pull-down and gel migration assays to study protein-protein and protein-nucleic acid interactions.

5. Unnatural amino acids can be inserted at specific sites. The insertion of unnatural amino acids at specific sites is of great significance for the development of recombinant protein drugs.

Principle of introduction of unnatural amino acids

6. Can be used to express granule-like viruses. This expression product has both the ability to elicit an immune response and no genetic material. Therefore, it cannot be copied. It is an ideal virus vaccine.

How to choose the right protein expression system ? please click here!

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