단백질 공학 Cell-free display systems for protein engineering (2)

Cell - Free Display에는 5가지 방법이 존재한다.

1. Alternantive to phage display and cell surface display systems

2. In vitro compartmentalization

3. Ribosome display

4. mRNA display

5. Covalent and noncovalent DNA display

4. mRNA display

Linkage of genotype and phenotype by mRNA

Similar with ribosome display

3' end of the mRNA template should be linked to a single stranded DNA bonded to puromycin -> covalent mRNA-protein fusion

과정

puromycin에 접착된 mRNA template가 vitro translation reaction 과정에서 사용되어 진다.
Protein 합성이후, puromycin은 ribosome을 들어가는데 이는 covalent mRNA - protein을 형성하기 위해서이다.

장점

covalent linkage between genotype and phenotype 은 ribsome display와 비교해서 거친 생화학적 상태에서 사용되어 질수 있다.

No bulky ribosome(ribosome은 binding을 방해하기도 하고 nonsepcific binding을 진행하기도 함.)

Very big libarary size ( ~10^14)

단점

Possible RNase contamination

Not stable at room temperature

Requires works at low temperatue -> RNAse에 민감하기 떄문이다.

5. Covalent and noncovalent DNA display

mRNA display를 토대로진행된다. P2A endonuclase fusion protein, M.Haelll fusion protein in compartements, AGT fusion protein in compartments 으로 총 3가지 방법이 존재하다.

(A) A DNA construct composed of the coding sequence for the protein of interest, the coding sequence for the DNA replication initiator protein RepA, the CIS element, and the R1 plasmid origin of replication (ori) is transcribed and translated in a standard, coupled in vitro reaction

(B) Biotinylated DNA encoding a fusion protein of streptavidin and a protein of interst is transcribed and translated in the aqueous phase of a water - in - oil emulsion. high affinity을 가지는 bio moelcule(Streptavidin)을 이용함.

(C) Streptavidin coated microbeads decorated with bitoinylated anti- tag antibodies and a single molecule of biotinylated DNA encoding a tagged protein are encapsulated in aqueous compartments (one bead per compartment), along with the reagents necessary for in vitro trascription and translation.