Production of proteins in cell-free system

1. Production of proteins in cell-free system Group 5

2. Why “In vitro” ? • Great freedom in process design and control • Production of toxic proteins • Short duration and high productivity

3. The basic of cell-free protein synthesis

4. Continuous flow in the in vitro protein synthesis 1988, a new method had introduced into cell free protein synthesis system. The continuous flow. Which made protein productivity into minigram level. 1996, a reasearch group developed a new way to modify continuous flow into semicontinuous culture. In this way, cell extract can prevent important components lost in the continuous culture, like elongation factors, rna polymerase, ribosomes, and other things. And release something harmful, like acetate, lactase and inorganic phosphate. Reaction voluome : 120 μl Protein productiviti : 6mg/ml Dinalysis membrane

5. Why Batch? • When semicontinuous culture have made big breakthrough in protein synthesis. But batch culture were remain unmove. Althought fed-batch can improve the protein productivity in batch system. Swartz still want the simple batch system? A. Batch system is easier to operate and need no complex equipment. B. Batch system is easier to scale up than all the other system.

6. How to scale up? How to cost down? Cell free protein synthesis simple flow chart: Cell extraction pretreatment & component supplement Cell culture & extract cell component Protein synthesis Energy source Protein activity System supplement Swartz’s research made lots of efforts to scale up and cost down.

7. Cell extraction Cell extract is one of the component is the CFPS system. Usually use S30 cell extract. S30 extract: (這裡我會補上小畫家的圖)

8. Cell extraction Cell extraction is one of the key point in the CFPS , because cell extract provide tRNA , rRNA, DNA polymerase, and other things to support in vitro transcription/translation. And the method for extracting cell component was established in the 1960. It’s cost lots of times and money. And Swartz modified this protocol made it quickly and cheaper. Save 6 hour operation time and 70% treating cost

9. Energy source • ATP is the most important energy source • The quantity of ATP decide the protein productivity • how to regenerate ATP in the cell free system become the most important issue

10. Energy source In the reaction mixture, there will have NTPs to provide energy for system, and also have PEP (PANOx SP ) or pyruvate (Cytomin) to regenerate ATP. Because PEP is expensive , so they try to use pyruvate . And using endogenesis enzyme to oxidize pyruvate into high energy compound.

11. Energy source Where PEP nor pyruvate, they just can regenerate ATP at 1:1 ration. But in the glycolytic, 1 glucose can regenerate two ATP. In order to that, Swartz try to use glucose as the major energy source , and succeed.

12. Energy source Further more Swartz wanted to replace NTPs with NMPs , because there should have enzymes can recover NMP to NTP.

13. Energy source Summary of the cost down processing These cost down processing after all, make a great economic benefit.

14. Scale up Batch volume of protein synthesis was limited at μL level (15μL) for a long time. Althought using PANOx SP system (PEP as second energy) can easier scale up in tube but Cytomin system cauldn’t scale up in the tube.

15. Scale up And Swartz thought productivity decrease may caused by oxygen shortage, so they transmit reaction from tube to flat film. To expanded gas /liquid intersurface. And made good effect.

16. The basis: PANOs system Conventional energy source: PEP, pyruvate PEP is expensive, and accumulation of the byproduct, phosphate, interfere protein synthesis Utilizing pyruvate has low production yields Solution: From substrate level phosphorylation to oxidative phosphorylation, which occur in organism A more natural chemical environment would encourage more natural metabolism

17. Mimicking E. coli cytoplasmic condition for efficient energy regeneration Objective: to alter elements of the in vitro system to better mimic the cell’s cytoplasm in the hope of increasing protein production yields from pyruvate. HEPES (unnatural components, is used out of its buffering range in cell-free system) , PEG (this polymer may negatively affect properties of the extract that are desirable for re-creating the in vivo environment), ionic solutes (Acetate, which may be detrimental for protein synthesis. Phosphate, is the by product interfering protein synthesis)

18. The approaches to mimic E. coli cytoplasmic condition Replace PEP with pyruvate Replace acetate with glutamate, but still use PEP as energy source Remove HEPES and replace PEG with spermidine and putrescine Remove: PEG HEPES Reduce: Acetate Ammonium Phosphate

19. Removing PEG

20. Magnesium concentrationre-optimization The magnesium concentration was reoptimized because of the higher affinity of PEP for magnesium relative to pyruvate and also because no significant phosphate accumulation was expected

21. Reducing Pi accumulation

22. Total yield of PANOx-SP Total yield of Cytomim system Soluble and active amounts of cytomim production Total yield of PANOx

23. Another approach: glucose • Least inexpensive commercial substrate • Natural carbon source of E.coli • Change the buffer • Optimize phosphate concentration • From PEP to G6P to glucose • Oxidative phosphorylation

24. Replace HEPES with Bis-Tris to make G6P or glucose energy system work Addition of phosphate increases CAT production when glucose is used as energy source

25. The most efficient way to produce ATP in an organism: oxidative phosphorylation 14C-glucose glucose (black), pyruvate (diagonal hatches), lactate (white), acetate (vertical lines), other (gray).

27. How to improve the protein folding?

28. Sulfhydral redox potential control • Disulfide bond formation requires arelatively oxidized enviroment. [4 mM] [1 mM] Stabilizing the sulfydral redox potential

29. Adding DsbC • Periplasmic disulfide bond isomerase • Require free sulfhydryls for activity

30. More natural environment • PEG: stabilizing mRNA • Spermidine and putrescine: improving the extent and fidelity of translation

31. Flow chart of modified method for enhancing disulfide bond formation S30 extract Mixed with 1 mM IAM for 30 min at RT 4 mM GSSG, 1 mM GSH 75 μg/ml DsbC 1.5 mM spermidine, 1 mM putrescine 300 μg/ml Skp Adding to the reaction mixture Template DNA addition Incubate for 3 hr

32. Synthesis of proteins containing non-nature amino acids • Methanococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) and tRNATyr (o-tRNA) OMe pAc pAz