Cell-free expression systems

1. Harini Chandra Affiliations Cell-free expression systems Cell-free systems make use of template DNA in the form of plasmids or PCR products to carry out in vitro transcription and translation. Protein microarray generation using cell-free expression systems eliminate the need for protein purification.

2. Master Layout (Part 1) 1 This animation consists of 4 parts: Part 1 – Commonly used cell-free systems Part 2 – E. coli S30 extract Part 3 – Wheat germ extract Part 4 – In vitro protein synthesis Cell-free expression systems 2 3 Rabbit reticulocyte lysate (RRL) Wheat germ extract (WGE) E. coli S30 4 5

3. Definitions of the components:Part 1 – Commonly used cell-free systems 1 1. Cell-free expression systems: Cell-free systems are used for in vitro protein synthesis starting from DNA templates. These systems contain all the necessary components and machinery for transcription and translation. Some factors such as energy generating components, essential amino acids etc. need to be added to the system for successful protein synthesis. 2. E. coli S30: This is a commonly used bacterial expression system that is capable of producing protein yields of around ~6 mg/mL (Jackson et al., 2004). This system however is not capable of carrying out post-translational modifications (PTMs) of proteins due to the absence of required machinery for this process and very often produces incomplete protein chains. DNA templates obtained from bacterial sources are commonly used with this cell-free lysate. 3. Rabbit reticulocyte lysate (RRL): A mammalian cell-free system that also gives protein yields of ~6 mg/mL (Jackson et al., 2004). This system is more suitable for expression of full length eukaryotic proteins from plant and animal sources that require proper folding and PTMs. 4. Wheat germ extract (WGE): This is a cell-free expression system that is capable of producing full length proteins with correct folding and PTMs from bacterial, plant or animal sources. Yields obtained in this system are however slightly lower than the E. coli and RRL. 2 3 4 5

4. Part 1, Step 1: 1 Cell-free expression systems 2 Rabbit reticulocyte lysate (RRL) Wheat germ extract (WGE) E. coli S30 3 4 Action Description of the action Audio Narration Each of the headings must appear sequentially followed by highlighting of only the first two as shown. (Please redraw all figures.) First show the main heading on top appearing followed by each of the sub-headings appearing in sequence along with their respective figures. The user should be allowed to click on any of the three to obtain the details as explained in the previous slide. Finally, the first two must be highlighted as shown. As given in the previous slide. To be shown when the user selects one of the titles. 5

5. Master Layout (Part 2) 1 This animation consists of 4 parts: Part 1 – Commonly used cell-free systems Part 2 – E. coli S30 extract Part 3 – Wheat germ extract (WGE) Part 4 – In vitro protein synthesis Cell lysis & centrifugation Extraction of cell-free system 2 Nucleoid 3 Active E. coli cell Enzymes 4 Transcription Translation Ribosomes mRNA Circular DNA Expressed proteins 5 1. Schwarz, D., Dotsch, V., Bernhard, F. Production of membrane proteins using cell-free expression systems. Proteomics 2008, 8, 3933-3946. 2. Biochemistry by A.L.Lehninger, 3rd edition (ebook)

6. Definitions of the components:Part 2 – E. coli S30 extract 1 1. Active E. coli cell:E. coli is possibly the most studied prokaryotic cell and is easily available. The active state of this bacterium or any other cell implies that it is in the process of synthesizing its proteins required for various cellular functions. In this state, the cell produces various enzymes and other factors in its cytoplasm that are required for the processes of transcription and translation. 2. Nucleoid: The genetic material of the cell is present in its nucleoid in the form of a single, circular molecule of DNA. Other circular DNA segments known as plasmids may also be present in the cytoplasm. 3. Circular DNA: The genetic material of the E. coli cell that codes for the various proteins required for cellular functioning and is responsible for transmission of characteristics from one generation to the next. 4. Enzymes: Biochemical catalysts that increase the rate of specific reactions by as much as million fold. The various enzymes involved in the process of transcription and translation such as RNA polymerase, peptidyl transferase, amino acyl tRNA synthetase etc. are present in the cell-free systems. 5. Transcription: The process by which template DNA is converted into its complimentary mRNA sequence, which acts as the adaptor molecule between DNA and proteins. 2 3 4 5

7. Definitions of the components:Part 2 – E. coli S30 extract 1 6. mRNA: The single stranded messenger RNA molecule that acts as the link between the base sequence of DNA and the amino acid sequence of proteins. 7. Ribosomes: These are complex and intricate cellular machines that efficiently read the base sequence of the mRNA as triplet codons and incorporate the corresponding amino acid into the growing polypeptide chain. 8. Translation: The process by which the mRNA in converted into its corresponding amino acid sequence. 9. Expressed proteins: The proteins that are synthesized by the cellular transcription and translation machinery which are required for various functions of the cell. 10. Extraction of cell-free system: The actively growing and functioning cells are lysed and then centrifuged at 30,000g to collect the supernatant containing cell-free extract. This has all the necessary components for in vitro protein synthesis. 2 3 4 5

8. Part 2, Step 1: 1 Nucleoid 2 N N N Active E. coli cell Essential amino acids N Enzymes N N 3 ATP ATP Transcription Translation Ribosomes mRNA ATP Circular DNA ATP Expressed proteins Nucleotides ATP 4 Action Description of the action Audio Narration The cell structure must be zoomed into and the animation below must be shown to be taking place. Actively growing and replicating E. coli cells can be used for extracting cell-free lysates. These cells that are in the process of growth and division are constantly producing proteins and other factors required for the various cellular processes. Co-factors and enzymes such as RNA Polymerase, peptidyl transferase etc are available in significant quantities due to cellular processes of transcription and translation taking place in the cell. (Please redraw all figures.) First show the cell structure with its labels. This must be zoomed into and several of the figures shown in the box below must be shown. The 3 colored circles must then move in a circle around the multi-colored circle (DNA) followed by appearance of the colored strands and the brown shapes on top of them. These must move across the strand as depicted and the colored figures on the right must then appear. 5

9. Part 2, Step 2: 1 Isolation of cell-free extract Centrifugation 2 Lysis buffer Supernatant – cell free extract 3 Pellet – lysed cells Active E. coli cells 4 Action Description of the action Audio Narration The cells are lysed with a suitable buffer and then centrifuged at 30,000g to collect the supernatant containing the extract. Lysate that is extracted from such actively growing and dividing cells will contain all required cellular machinery to carry out in vitro protein synthesis and requires addition of essential amino acids, nucleotides, salts and other energy generating factors. The tube must be shown to enter the instrument shown and then removed. (Please redraw all figures.) First show the tube with the particles as depicted. Then show the tube being filled with the blue solution and then being placed in the instrument. The lid must then be closed and shown closed for a few seconds after which the lid must be opened and the tube taken out to show the solution as shown on the right. 5

10. Master Layout (Part 3) 1 This animation consists of 4 parts: Part 1 – Commonly used cell-free systems Part 2 – E. coli S30 extract Part 3 – Wheat germ extract (WGE) Part 4 – In vitro protein synthesis Wheat seeds 2 Processing steps Clean wheat embryo Selection & washing Wheat embryo 3 Extraction, fractionation & purification Bran Endosperm Germ/embryo 4 Wheat germ extract 5 Takai, K., Sawasaki, T., Endo, Y. Practical cell-free protein synthesis system using purified wheat embryos. Nat Protoc. 2010, 5 (2), 227-238.

11. Definitions of the components:Part 3 – Wheat germ extract (WGE) 1 1. Wheat seeds: Seeds from the wheat grain are used for extraction of the desired eukaryotic cell-free lysate. The structural components of the wheat seed include: a) Bran: This is the protective outer coating of the seed that is rich in fibres, trace minerals and B vitamins. b) Endosperm: The endosperm is the tissue that is produced surrounding the embryo around the time of fertilization and provides the seed with nutrition in the form of carbohydrates and proteins. It is this endosperm that is the source of nutrients in processed wheat foods like bread. c) Germ/embryo: The reproductive part of a cereal that grows into a plant and is a rich, concentrated source of several essential nutrients like folate, Vitamin E, trace metals etc is known as the germ or embryo. It is this part that contains the necessary cellular machinery and is therefore used for preparation of the extract. 5. Processing steps: The seeds are processed in order to reach the embryo by grinding them mildly followed by sieving to remove the outer seed-coat fragments. The embryos are finally separated by addition of organic solvents like cyclohexane or carbon tetrachloride for a short while which cause the embryos to float thereby allowing them to be separated and dried. 2 3 4 5

12. Definitions of the components:Part 3 – Wheat germ extract (WGE) 1 6. Selection & washing: The obtained embryos are then carefully screened to select only the good quality embryos that do not contain the outer endosperm coating. These selected embryos are washed thoroughly in cold water until any outer coating that may be present is removed. The endosperm coating contains inhibitors of protein synthesis and therefore must be removed. 7. Clean wheat embryo: The embryo that is free of any outer endosperm coating and is ready for further processing. 8. Extraction, fractionation & purification: Cleaned embryos are grinded with the extraction buffer followed by centrifugation at 30,000g at 4oC to separate the various components. The supernatant containing the lysate is removed and purified by chromatography to separate any components of the extraction buffer that are present. 9. Wheat germ extract: The desired, pure wheat germ extract is obtained for in vitro protein synthesis. 2 3 4 5

13. Part 3, Step 1: 1 Larger outer coat fragments Sieving Wheat seeds Grinding 2 Smaller particles 3 Bran Endosperm Separated embryos Germ/embryo Flotation 4 Action Description of the action Audio Narration As shown in animation. (Please redraw all figures.) First show the brown seeds, which must be zoomed into to depict the figure below. It must then be zoomed out and shown to enter the grey grinding machine. A powdery mass must come out of it and must be shown to pass through a mesh as shown on the right top. The fine particles coming out of this mesh must then be shown in a blue liquid with small particles on top and larger particles settling down. One of the most commonly used eukaryotic cell-free expression systems is obtained from the embryo of the wheat seeds. The seeds are grinded and then sieved to remove their outer coating fragments. The embryos and other small particles are floated in an organic solvent like cyclohexane. The floating embryos are quickly removed and dried to avoid any damage from the organic solvent. 5

14. Part 3, Step 2: 1 Extraction buffer 2 Grinding & extraction Clean embryos 3 Selected embryos washed 4 Action Description of the action Audio Narration The dried embryos are then carefully sorted such that only the good embryos without any endosperm coating are selected. The endosperm contains certain inhibitors of protein synthesis that must be removed. The selected embryos are washed thoroughly with cold water after which they are mixed with the extraction buffer and grinded. Particles shown on left must be washed with water and then mixed with the solution on the right. First show the particles on the left followed by the blue solution appearing over it. The blue solution must then be poured out and this procedure must be repeated three times followed by appearance of the grey particles in the middle panel. This is followed by addition of these particles into the blue solution in the container, The particles must be shown to break into several smaller fragments when this happens. 5

15. Part 3, Step 3: 1 Centrifugation 2 Wheat germ extract 3 4 Action Description of the action Audio Narration This solution must be centrifuged at 30,000g at 4oC which results in the wheat germ extract forming a layer in between the top fatty layer fraction and the pellet at the bottom. This fraction can be separated and then purified by chromatographic methods to remove any components of the extraction buffer. This cell free lysate is capable of synthesizing full length eukaryotic proteins with yields of around 4 mg/mL. As shown in animation. (Please redraw all figures.) First show the solution in the tube with the particles at the bottom. This tube must then enter the instrument shown and the lid must be closed. It must then be opened after a couple of seconds and the tube must be removed from the instrument to show the solution as shown on the right. 5

16. Master Layout (Part 4) 1 This animation consists of 4 parts: Part 1 – Commonly used cell-free systems Part 2 – E. coli S30 extract Part 3 – Wheat germ extract (WGE) Part 4 – In vitro protein synthesis DNA template N N N 2 N N N Termination sequence Ribosome binding site Gene of interest Promoter sequence 3 Exogenous factors Cell-free lysate Essential amino acids ATP tRNA Enzymes Ribosomes 4 ATP ATP Nucleotides 5

17. Definitions of the components:Part 4 – In vitro protein synthesis 1 1. DNA template: The sequence coding for the protein of interest which must be transcribed and translated using the suitable expression system. This DNA may either be in the form of PCR DNA or plasmid DNA. 2. Promoter sequence: The region of DNA located upstream of the gene of interest and having a specific base sequence that facilitates transcription by binding to the sigma subunit of RNA polymerase enzyme. Commonly used promoters include T7, SP3 or T3. 3. Ribosome binding site: For translation to take place from the synthesized mRNA, a translation initiation site is required which will allow the ribosome and other factors required for the process to bind to it. Translation initiation sequences such as the Shine-Dalgarno for prokaryotes and Kozak sequence for eukaryotes are added for in vitro protein synthesis. 4. Gene of interest: The gene that codes for the desired protein or polypeptide target. This may be obtained through PCR amplification or may be present in the form of a plasmid. 5. Termination sequence: For transcription and translation to be appropriately halted, termination sequences must be present so that the desired protein is correctly synthesized. 2 3 4 5

18. Definitions of the components:Part 4 – In vitro protein synthesis 1 6. Cell-free lysate: As described in Part 1, the cell-free lysate contains all the necessary cellular machinery for protein synthesis to take place. The lysate is extracted from an actively growing cell and therefore contains enzymes, ribosomes, tRNA molecules, non-essential amino acids etc. Mammalian cell-free lysates like wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL) allow post translational modifications to occur in the protein while bacterial lysates like E. coli extract do not possess the required enzymes for this process. 7. Exogenous factors: In addition to the cell-free lysate, certain exogenous factors like essential amino acids, nucleotides, energy generating factors etc. must be added to the system for the process to take place successfully. 2 3 4 5

19. Part 4, Step 1: 1 DNA template stored on ice which must have the following components 2 3 Ribosome binding site Termination sequence Promoter sequence Gene of interest 4 Action Description of the action Audio Narration Zoom into the tube shown on top and then show each region being highlighted as shown in the animation. The DNA template is thawed and then placed on ice during the preparatory process. For in vitro protein synthesis to take place, the DNA template must contain the gene coding for the protein of interest. In addition to this, there must be a promoter sequence which can initiate the transcription process, a translation initiation sequence for binding of the ribosome as well as suitable termination sequences to correctly synthesize only the protein of interest. (Please redraw all figures.) First show the tube being placed in the vessel on top with cubes of ice. The tube must be zoomed into and then show the DNA template in the grey scale and then appearance of each region starting from the left. As the label for each region appears, that region must be highlighted as shown in the animation. 5

20. Part 4, Step 2: 1 Essential amino acids ATP ATP ATP 2 N N N N Nucleotides N N 3 Cell-free lysate Exogenous factors DNA template stored on ice tRNA 4 Enzymes Ribosome Action Description of the action Audio Narration The thawed cell-free lysate containing the essential cellular machinery for protein synthesis is added to the DNA template followed by the other exogenous factors that are required for the process. All these are done while storing the template on ice to ensure that there is no loss of activity. As shown in animation. (Please redraw all figures.) First show the tube stored in the vessel on ice followed by appearance of the blue tube which must be zoomed into and the blue cloud must be shown. It must be zoomed out and then hand must be shown to add solution to the grey tube as depicted. Next the orange tube must appear. Again, it must be zoomed into and the orange cloud must be shown followed by zooming out and the hand motion adding solution to the grey tube. Color of the solution in grey tube must change upon addition. 5

21. Part 4, Step 3: 1 30oC ATP DNA template 2 Incubate at 30oC Enzymes N N N N N N N N ATP Nucleotides N N N N 3 Cell-free lysate ATP tRNA ATP Essential amino acids ATP mRNA Ribosomes 4 Action Description of the action Audio Narration The tube containing all the required components is then incubated at 30oC. Enzymes for transcription bind to the promoter sequences and in the presence of other factors like ATP and nucleotides, they carry out synthesis of the mRNA transcript. This mRNA is then translated into the corresponding protein with the help of ribosomes, tRNA, enzymes and other factors required for the process. (Please redraw all figures.) First show the tube being placed in the box on top and then zoom into the tube. In that, show ‘DNA template’ followed by the cloud on top along with the colored circles and their respective labels. Then show the green ‘ATP’ circles and the other chemical structures. The colored circles must then move as shown and must result in the ‘mRNA’ structure appearing below followed by the cloud and all other components shown. The brown shapes must then move as depicted. As shown in animation. 5

22. Interactivity option 1:Step No:1 1 Plant, animal & bacterial Full length Full length Plant, animal & bacterial Bacterial Incomplete 2 3 ~6 mg/mL Possible Not possible Possible ~6 mg/mL 4 ~4 mg/mL Options Results Interacativity Type Boundary/limits The correct answers are depicted in the animation above. At the end of every row, if the user has got it correct, a tick mark must appear at the side else a cross. Once the entire table is done correctly, a message saying ‘Great job’ must appear. Each row of the table must appear one at a time and three options to fill that row must appear along with it. User must drag and drop the options into the correct cell. Drag and drop. 5 Jackson, A. M., Boutell, J., Cooley, N., He, M. Cell-free protein synthesis for proteomics. Brief. Funct. Genom. Proteom. 2004, 2(4), 308-319.

23. Questionnaire 1 1. Which of the following components will not be present in the WGE lysate? Answers: a) Ribosomes b) RNA Polymerase c) Tryptophan d)‏ peptidyl transferase 2. Catalytic inhibitors of protein synthesis are found in which component of wheat seeds? Answers: a) Embryo b) Endosperm c) Bran d)‏ Germ 3. Which of the following systems would be preferred for producing high yields of a full length eukaryotic protein? Answers: a) E. coli S30 b) WGE c) Xenopus oocyte d)‏ RRL 4. The function of the Shine Dalgarno sequence is Answers: a) Binding of RNA Polymerase b) Binding of sigma subunit c) Binding of tRNA d)‏ Binding of ribosome 5. Which of the following is not a promoter sequence? Answers:a) Kozak b) T7 c) SP3 d)‏ T3 2 3 4 5

24. Links for further reading Reference websites: http://www.promega.com/applications/prtn_exp/expression.htm Research papers: • Schwarz, D., Dotsch, V., Bernhard, F. Production of membrane proteins using cell-free expression systems. Proteomics 2008, 8, 3933-3946. • Mikami, S., Masutani, M., Sonenberg, N., Yokoyama, S., Imataka, H. An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expression & Purification 2006, 46, 348-357. • Katzen, F., Chang, G., Kudlicki, W. The past, present and future of cell-free protein synthesis. Trends Biotechnol. 2005, 23 (3), 150-156. • Jackson, A. M., Boutell, J., Cooley, N., He, M. Cell-free protein synthesis for proteomics. Brief. Funct. Genom. Proteom. 2004, 2(4), 308-319.