Cell-free Systems for Recombinant Protein

Productionand for 15N/13C Labeled

Protein Production for NMR Studies

Cell-free (CF) protein synthesis provides a recently developed and powerful alternative tool for protein production

Cell-free protein synthesis

Translation SystemsTranslation Systems

cell-free protein synthesis provides a completely open system

Linked Transcription:TranslationLinked Transcription:Translation Coupled Transcription:TranslationCoupled Transcription:Translation

Translation SystemsTranslation Systems

"Linked" and "coupled" systems use DNA as a template. RNA is transcribed from the DNA and subsequently translated without any purification. Such systems typically combine a prokaryotic phage RNA polymerase and promoter (T7, T3, or SP6) with eukaryotic or prokaryotic extracts to synthesize proteins from exogenous DNA templates. DNA templates for transcription:translation reactions may be cloned into plasmid vectors or generated by PCR

"Linked" And "Coupled" Transcription:Translation Systems

Primer Sequences for PCR-generated Translation Templates

DNA templates for translation using "coupled" or "linked" transcription:translation systems can be easily generated by PCR. Below are the upstream (5')primer sequences to produce PCR products for T7-driven transcription and subsequent translation in a retic lysate and E.coli extract, respectively.

                                                            

Because the transcription and translation reactions are separate, each can be optimized to ensure that both are functioning at their full potential.

This bacterial translation system gives efficient expression of gene products in a short amount of time.

Gene Of Interest = GOI

•Toxic proteins and proteins containing non natural amino acids can be made efficiently

•Proteins forming inclusion bodies in vivo systems

•The reaction is fast (proteins that are sensitive to proteolytic degradation)

•The reaction can be carried out in small volumes (materials are used more efficiently and economically)

•Many of the enzymatic activities present in live cells are suppressed

Advantages of cell-free protein synthesis

The reaction is independent of cell growth:

Preparation of cell-free extract

•E.coli cells

•Wheat germ

•Rabbit reticulocytes

The most frequently used cell-free translation systems consist of extracts from :

E. coli BL21(DE3)

BL21 (DE3) pLysS

Rosetta (DE3) pRare

BL21 Star (DE3)

A19

Source for S30 E. coli lysates:

Fermenter

French Press cell disruption device

Dialysis membranes (15 kDa)

S30 extract preparation procedure:

In principle, it should be possible to prepare a cell-free extract for in vitro translation of mRNAs from any type of cells. In practice, only a few cell-free systems have been developed for in vitro protein synthesis. In general, these systems are derived from cells engaged in a high rate of protein synthesis.

In vivo, reticulocytes are highly specialized cells primarily responsible for the synthesis of hemoglobin, which represents more than 90% of the protein made in the reticulocyte

PEP = phosphoenolpyruvate

All are prepared as crude extracts containing all the macromolecular components (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNA.

To ensure efficient translation, each extract must be supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. coli lysate), and other co-factors (Mg2+, K+, etc.).

What cell-free extract contains?

1. PEP system

2. CP system

Provide all the high molecular weight components of the translation machinery

Ribosomes

Translation factors

Amino-acyl-tRNA synthetases

Methionyl-tRNA transformylase (needed for initiator Met-tRNA)

To ensure efficient translation, each extract must be supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase)

Preparation of cell-free extract

Formylation in protein synthesis

In bacteria and organelles, the initiation of protein synthesis is signaled by the formation of formyl-methionyl-tRNA ((f-Met)-tRNA).

10-formyltetrahydrofolate (f-Met)-tRNA (Met)-tRNA

ARSEs= aminoacyl-tRNA synthetases

Configuration and productivity of cell-free systems

•Rapid depletion of precursors

•Accumulation of inhibitory products

•The reaction times are

extended up to approx. 2 h

Continuous-exchange cell-free (CECF) system

•Supply of fresh precursors

•Continuous removal of deleterious reaction by-products

•The reaction times are

extended up to approx. 16 h

First generation CF expression systems

Reaction conditions of E. coli cell-free systems

CF expression can be performed in small analytical scale reactions with approximately 200l RM for optimization reactions and in larger preparative scale reactions of 1–2ml RM for the production of protein.

Reaction mixture

Feeding mixture

The reaction has to be incubated with intensive agitation at 37°C

HEPES

DTTATP

CTP, GTP, UTP cAMP

Folinic acidNH4 acetate

K glutamate

Creatine phosphate

Creatine kinaseAmino acidMg acetate

tRNAS30 Extract

DNA plasmidT7 RNAP o T7

plasmid

Reaction mixture

HEPES

DTTATP

CTP, GTP, UTP cAMP

Folinic acidNH4 acetate

K glutamate

Creatine phosphate

Creatine kinaseAmino acidMg acetate

Feeding mixture

Spectra/Por DispoDialyzer

Design of DNA templates for cell-free systems

The transcription in E. coli coupled transcription/translation CF systems is operated by the phage T7-RNA polymerase.

The purified enzyme has to be added into the RM 100g/ml

rbsrbs

The plasmid coding T7-RNA polymerase has to be added into the RM 30g/ml

AUTOINDUCTION SYSTEM

Linear DNA as a template for cell-free systems

The possibility to use linear templates generated by PCR in the CF-system eliminates time consuming cloning/subcloning steps and allows the rapid screening of a variety of expression constructs (mutants)

High degradation by exonucleases present in the E. coli extracts

templates cyclize by the endogenous ligase activity of E. coli S30 extracts

single-stranded overhang

single-stranded overhang

Example of E. coli cell-free systems

200 l reactions mixture

M h h

PpiB

T7RNAPOL pKO1166

M hhhhh

bio-14k

200 l reactions mixture

pKO1166

Cell-free systems of 15N-labeled proteins for NMR studies

In cell-free expression the target protein is the only protein synthesized and the reaction can be carried out in small volumes

Isotope-labelled starting materials are used more efficiently and economically than for conventional in vivo labelling methods

15N-labeled proteins can be analyzed by NMR spectroscopy of the crude reaction mixture without chromatographic separation or concentration

15N-Gly 15N-Arg15N-Protein

An attractive application for this method is the production of selectively isotope-labelled samples

cell-free systems of selectively 15N amino acid labelling

for NMR studies

Metabolic enzymes present in the S30 extract can interconvert amino acids, leading to scrambling of 15N labels, and also their incorporation into metabolic by-products

transaminase transaminase activityactivity

Heat treatment of S30 extract

Addition of chemicals

Enzymatic activities in cell-free extract

cell-free systems and incorporation of non-natural amino acids

•incorporation of fluoro-tryptophan incorporation of fluoro-tryptophan

19F-NMR offers a sensitive way of determining whether a protein is folded or unfolded without prior purification of the protein

•incorporation of selenomethionineincorporation of selenomethionine

The incorporation of heavy atoms such as Selenium helps solving the phase problem in X-ray crystallography using multi-wavelength anomalous diffraction (MAD)

cell-free systems of membrane proteins

CF protein synthesis allows the production of membrane proteins in two very different modes:

•As precipitate

•As soluble protein (detergents)

•The precipitated MPs are harvested from the RM by centrifugation•The pellet is washed for several times in an appropriate buffer (e.g. 15 mM sodium phosphate, pH 6.8, 1 mM DTT) to remove the impurities•The pellet is washed with a detergent that has poor solubilization properties (e.g. 3% n-octyl-β-glucopyranoside (β-OG)) to remove the impurities•The pellet is solubilized in a mild detergents buffer (e.g.

2% 1-myristoyl-2 hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG)). •Incubation on a shaker at 30 °C for one hour is usually sufficient for the quantitative solubilization.

MP precipitates are structurally different from inclusion bodies

The efficiency of solubilization certainly depends on the specific recombinant MP as well as on the type of detergent.

cell-free systems of membrane proteins as precipitate

•Defined amounts of detergents are added directly into the reaction •The proteins are embedded immediately into preformed detergent micelles in a soluble form. •Soluble protein fractions are separated from precipitates after the reaction by centrifugation at 20,000g for 30 min at room temperature. • Proteomicelles could be purified directly out of the RM and critical steps like the destabilization and isolation of MPs from membranes are eliminated.

The supplied detergent must be tolerated by the CF system

cell-free systems of membrane proteins in soluble form

The type of detergent and its concentrations (CMC) must be subjected to optimization