Cell-free Systems for Recombinant Protein Production
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Transcript of Cell-free Systems for Recombinant Protein Production
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