단백질 공학 Cell-free display systems for protein engineering (1)

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

5가지 중에서 (1)~ (3)을 정리하고자 한다. 

 

목차

 

1. In Vitro Proetin Synthesis Reaction

3가지 필수구성요소( Cell extract, Templates, Additives to enhance protein synthesis efficiency)가 존재한다.

 

Extract(추출물)

고려되어야할 사항들 : efficiency of protein synthesis, post-traslation modification, difficulty in protein folding, cost, time

 

Extract Preparation(추출 준비)

S30 extract : 두번의 원심분리를 진행하여 용해되지 않는 물질들을 제거한다.(활성을 잃었기 때문) 그리고 soluble fraction containing major macromolecules을 recover한다.

Degradation of endogenous mRNA by incubating at room temperature

Display agains an appropriate buffer

Freezing

 

Additives

Protein sysnthesis의 효율성을 향상시키딘다.

Amino acids, Key ions, tRNAs..이 이용된다.

Solubility를 향상시키기위해 Lipids 와 세제를 첨가한다.

 

Template

Exogenous mRNA 또는 DNA

5' cap and 3' poly(A) tail for eukaryotic systems - mRNA에서 capping이 일어남

capping을 하는 이유는 Exonucleases를 보호하기 위해서이다. 출처 : 위키미디아

 

Reaction Conditions

온도와 시간을 최적화 시켜주어야한다.

높은 온도 : 반응이 빠르게 나타나지만 Proteins이 dnature한다.

긴 incubation time : higher chances of degradation of produced proteins

 

 

Uncouple/Coupled systems

Uncoupled system : transcription - separation of mRNA - traslation

Coupled system : simultaneous transcription/translation in the smae media (DNA, RNA polymerase, and NTPs to produce mRNA)

 

 

The PURE(Protein Synthesis using Recombinant Elements) System

All recombinant translation factors except the ribosome and tRNAs purified from E.coli lysates

장점 : Defined system compared to the lysate based system -> minimize nuclease and proteases

단점 : Low yeild compared to the lysate based system (효율적이지는 않지만 통제가 가능)

 

 

Cell-Free Protein Synthesis for Directed Evolution

Genotype-Phenotype Linkage, Randomization of genes(다양성 부여), Selection or Screening(선별), Isolation of clones for a desired phenotype(원하는 clone을 isolation 가능)

 

Directed Evolution Using Cell - Free Display System

 

2. Type of Vitro Selection or screen - In vitro Compartmentalization

1. Binding based selecitons (ribosome display, mRNA display, DNA display)

used for engineering proteins with improved affinity, stablilty, and folding - (affinity, stabitliy, folding 등 특성에 따라 engineering이 가능하다.)

 

Binding of the displayed library to immobilized ligands

Washing (staringency should be optimized)

Recovery of thightly bound members

PCR or RT- PCR

Next round screening

Enrichment after multiple rounds screening

Enrichment after multiple reounds screeing

Function or sequence analysis for isolated individual members

 

2. In vitro compartmentalization(IVC, 인위적으로 하는 시스템..)

mainly used for enzyme evolution coupled with FACS

Compartmentalization 이후 FACS를 하기위해서 bead에 display가 붙어있어야한다.

Fluorescent substrates can diffuse in/out

Isolation of variants with a high enzymatic activity

 

Types of Genotype - Phenotype Linkages

Ribosome display : C - terminal fusion for appropriate distance of generated polypeptides from the ribosome and suitable protein folding (polypeptides 가 ribosome 에서 만들어진 상태를 유지시킴)

mRNA display : DNA - puromycine linker at C - Terminus

DNA display : N or C - terminal fusion for binding to DNA

In vitro compartmentalization : N or C - termninal fusion for tethering to microsphers

 

Choice of Transalation System

Ribosome display system :

Coupled(translation과 transcription을 동시 진행)/uncoupled(translation 이후 transcription 진행) in vitro expresion systems 가능

For precise control of mRNA level, uncoupled reaction is better ( 정확한 통제를 위해서는 uncoupled 방식이 더 좋음)

 

mRNA display system :

chemical modifiation of mRNA template after transcription을 요구 (transcription 이후에 modification이 진행된다.)

Should be done with uncoupled in vitro systmes (Translation만 일어나야 하기 때문에 uncoupled 방식으로 진행)

 

In Vitro Display의 장/단점

장점 :

Large library size ( ~up to 10^14)

Easy display of toxic proteins

Straightforward randomization techniques (not necessary introduction to the cells)

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단점 :

Problems in protein folding (단백질의 접힘X)

Post - translational modificaton (박테리아를 이용하면 일어나지 않는다.)

Not quantitative in screening for the affinity based in vitro systems (FACS를 사용하지 않기 때문에 quantitative 하지 않는다.)

 

3. Ribosome Display

mRNA와 protein을 연결하는 방법 : Physical linkage between mRNA(genotype) and protein(phenotype) by ribosome complex

Transcription of dsDNA to mRNA

Translation for a short time (10~15 min)

Dilution(희석) in cold buffer with high(고농도) Mg2+ for crosslink the phospahte groups and inhibiting the dissociation of the ternary(mRNA-Protein-ribosome) complexes -> Translation time should be potimized to get enough production time/low degradation of mRNA

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Binding to the target immobilized to solid surface

Washing and recovery of mRNA

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Bigger library size는 phage display와 비교되기도 한다.

Successfully used to isolated affinity matured scFV antibody fragments and scaffold proteins

Used to evolve enzymes to increase catalytic activity of enzymes

Used to evolve folding properties

 

Ribosome display의 장/단점

장점

The first fully in vitro display technique

큰 사이즈가 가능하다.(10^12 - 10^14)

Ribosome은 단백질의 solubitily를 향상시킨다. 그리고 침전되지 않는다.

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단점

Some intrinsic affinity of ribosome or mRNA against the target

RNA molecules(RNA aptamer) can be evolved by SELEX(Systematic Evloution of Ligands by Exponential Enrichment) [functinal catatlytic RNA에 의해서 일어날수 있음]

Risk of RNase contamination -> RNase - free pipette tips, containersm and buffers

A special RNase-deficient strain (MRE600) can be used

Screening (Selction, washing and elution) should be done at low temperature to minimize RNase activity [RNase의 activity를 최소화시키기위해서 저온에서 진행되어야만함.]