Production of proteins in cell-free system

Group 5

Why “In vitro” ?

• Great freedom in process design and control

• Production of toxic proteins

• Short duration and high productivity

The basic of cell-free protein synthesis

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.

Dinalysis membrane

Reaction voluome : 120 μlProtein productiviti : 6mg/ml

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.

How to scale up? How to cost down?

Cell free protein synthesis simple flow chart:

Cell culture & extract cell component

Cell extraction pretreatment &

component supplement

Protein synthesis Energy source

Protein activity

System supplement

Swartz’s research made lots of efforts to scale up and cost down.

Cell extraction

Cell extract is one of the component is the CFPS system. Usually use S30 cell

extract.

S30 extract:

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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

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

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.

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.

Energy source

Further more Swartz wanted to replace NTPs with NMPs ,

because there should have enzymes can recover NMP to NTP.

Energy source

Summary of the cost down processing

These cost down processing after all, make a great economic benefit.

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.

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.

The basis: PANOs system

Conventional energy source: PEP, pyruvatePEP is expensive, and accumulation of the byproduct, phosphate, interfere protein synthesisUtilizing pyruvate has low production yields

Solution: From substrate level phosphorylation to oxidative phosphorylation, which occur in organismA more natural chemical environment would encourage more natural metabolism

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)

The approaches to mimic E. coli cytoplasmic conditionReplace acetate with glutamate, but still use PEP as energy source

Replace PEP with pyruvate

Remove HEPES and replace PEG with spermidine and putrescine

Remove:PEGHEPES

Reduce:AcetateAmmoniumPhosphate

Removing PEG

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

Reducing Pi accumulation

Total yield of PANOx-SPTotal yield of Cytomim system

Soluble and active amounts of cytomim production

Total yield of PANOx

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

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

The most efficient way to produce ATP in an organism: oxidative phosphorylation

glucose (black), pyruvate (diagonal hatches), lactate (white), acetate (vertical lines), other (gray).

14C-glucose

How to improve the protein folding?

Sulfhydral redox potential control

• Disulfide bond formation requires arelatively oxidized enviroment.

[4 mM] [1 mM]

Stabilizing the sulfydral redox potential

Adding DsbC

• Periplasmic disulfide bond isomerase

• Require free sulfhydryls for activity

More natural environment

• PEG: stabilizing mRNA

• Spermidine and putrescine: improving the extent and fidelity of translation

S30 extract

Mixed with 1 mM IAM for 30 min at RT

4 mM GSSG, 1 mM GSH75 μg/ml DsbC

1.5 mM spermidine, 1 mM putrescine300 μg/ml Skp

Adding to the reaction mixture

Template DNA addition

Incubate for 3 hr

Flow chart of modified method for enhancing disulfide bond formation

Synthesis of proteins containing non-nature amino acids

• Methanococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) and tRNATyr (o-tRNA)

OMe pAc pAz