Cell-free Systems for Recombinant Protein Production

1. Cell-free Systems for Recombinant Protein Production and for 15N/13C Labeled Protein Production for NMR Studies

2. Cell-free protein synthesis Cell-free (CF) protein synthesis provides a recentlydeveloped and powerful alternative tool for protein production Translation Systems Linked Transcription:Translation Coupled Transcription:Translation cell-free protein synthesis provides a completely open system

3. Translation Systems

4. "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.

5. 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.

6. Gene Of Interest = GOI

7. Advantages of cell-free protein synthesis The reaction is independent of cell growth: • Toxic proteins and proteins containing nonnaturalamino acids can be made efficiently • Proteinsforming inclusion bodiesin vivo systems • The reaction is fast (proteins that are sensitive to proteolyticdegradation) • The reaction can be carried out in small volumes (materials are usedmore efficiently and economically) • Many of the enzymatic activities present in live cells are suppressed

8. 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. E. coli BL21(DE3) BL21 (DE3) pLysS BL21 Star (DE3) Rosetta (lDE3) pRare A19 Preparation of cell-free extract The most frequently used cell-free translation systems consist of extracts from : • E.coli cells • Wheat germ • Rabbit reticulocytes 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 Source for S30 E. coli lysates: S30 extract preparation procedure: Fermenter French Press cell disruption device Dialysis membranes (15 kDa)

9. PEP = phosphoenolpyruvate

10. 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?

11. 1. PEP system 2. CP system

12. Preparation of cell-free extract 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)

14. 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 (Met)-tRNA (f-Met)-tRNA

15. ARSEs= aminoacyl-tRNA synthetases

16. First generation CF expression systems Configuration and productivity of cell-free systems 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 • Rapid depletion of precursors • Accumulation of inhibitory products • The reaction times are • extended up to approx. 2 h

17. Reaction conditions of E. coli cell-free systems Spectra/Por DispoDialyzer The reaction has to be incubated with intensive agitation at 37°C Feeding mixture Reaction mixture Reaction mixture Feeding mixture HEPES DTT ATP CTP, GTP, UTP cAMP Folinic acid NH4 acetate K glutamate Creatine phosphate Creatine kinase Amino acid Mg acetate HEPES DTT ATP CTP, GTP, UTP cAMP Folinic acid NH4 acetate K glutamate Creatine phosphate Creatine kinase Amino acid Mg acetate tRNA S30 Extract DNA plasmid T7 RNAP o T7 plasmid CF expression can be performed in small analytical scale reactions with approximately 200ml RM for optimization reactions and in larger preparative scale reactions of 1–2ml RM for the production of protein.

18. rbs AUTOINDUCTION SYSTEM The plasmid coding T7-RNA polymerase has to be added into the RM 30mg/ml The purified enzyme has to be added into the RM 100mg/ml 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.

20. Linear DNA as a template for cell-free systems single-stranded overhang High degradation by exonucleases present in the E. coli extracts single-stranded overhang templates cyclize by the endogenous ligase activity of E. coli S30 extracts 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)

21. Example of E. coli cell-free systems M M 0h 1h 2h 4h 6h O/N 6h O/N 6h O/N T7RNAPOL pKO1166 pKO1166 bio-t14k PpiB 200 ml reactions mixture 200 ml reactions mixture

22. Cell-free systems of 15N-labeled proteins for NMR studies 15N-labeled proteins can be analyzed by NMRspectroscopy of the crude reaction mixture without chromatographicseparation or concentration 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 usedmore efficiently and economically than for conventionalin vivolabelling methods

23. cell-free systemsof selectively 15N amino acid labelling for NMR studies 15N-Protein 15N-Arg 15N-Gly Anattractive application for this method is the production ofselectively isotope-labelled samples

24. transaminase activity Heat treatment of S30 extract Addition of chemicals Enzymatic activitiesincell-free extract Metabolic enzymes present in the S30 extract can interconvertamino acids, leading to scrambling of 15N labels, and also their incorporation into metabolic by-products

25. cell-free systemsand incorporation of non-natural amino acids • 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 selenomethionine The incorporation of heavy atoms such as Selenium helps solving the phase problem in X-ray crystallography using multi-wavelength anomalous diffraction (MAD)

26. 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)

27. The efficiency of solubilization certainly depends on thespecific recombinant MP as well as on the type of detergent. cell-free systems of membrane proteins as precipitate MP precipitates are structurally different from inclusion bodies • 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-2hydroxy-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.

28. The type of detergent and its concentrations (CMC) must be subjected to optimization cell-free systems of membrane proteins in soluble form The supplied detergent must be tolerated by the CF system • 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.