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Protein ExpressionE.coli

How to prepare your own cell-free system

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Protocol for S30-ribosome-lysate
Preparation of the S30-lysate with expressed T7 RNA polymerase (optional)
Setting up a cell-free reaction
References
Necessary components

Here, we have a collection of protocols for the preparation of an E. coli based cell-free expression system in batch format.

It is interesting to note that the most expensive components of a cell-free reaction are the nucleotides which can be up to 70-80% of the total costs per reaction, if you prepare the reaction mix yourself. Therefore, it might be even advisable to use batch reactions only instead of dialysis reactions with a 10-20 fold excess of dialysis buffer containing all component except lysate, vector DNA, creatine kinase and tRNAs. Considering the fact that an optimum batch reaction yield up to 500 µg/ml within 2 hours, you would expect up to 5 mg/ml yield for a dialysis reaction with 1 ml internal and 10 ml external feeding solution within 20 hours.

Usually the increase by a factor of ~10 is rarely achieved in dialysis mode, thus you have less amount of total protein in the dialysis reaction than in 10 batch reactions which scale linearly as the reaction conditions are the same. In addition to that, you have a shorter incubation time and lower protein concentration which can be advantages for the solubility of the target protein.

The only disadvantages of the batch reaction is the need for more lysate (which is relatively cheap, if you prepare it yourself), more vector DNA (10 µg/ml) and a lower target protein concentration with more background protein from the lysate (which could cause a decrease in purity after purification).

At the moment, we don't offer cell-free expression as a service in our Core Facility but we can offer advice how to set it up in your own lab based on our earlier experience. Do not hesitate to contact us in case of questions or comments (pepcore@embl.de).

Protocol for S30-ribosome-lysate

(adapted from Kigawa et al. (2004), Preparation of Escherichia coli cell extract for highly productive cell-free protein expression, J. Struct. Fun. Gen., 5, 63-68)

E. coli strain: BL21(DE3), BL21(DE3) CodonPlus RIL or variants (not pLysS!)

! around 7 ml of S30-ribosome-lysate can be isolated from 1 litre of culture !

Per litre medium:

5.6 g KH2PO4    
28.9 g K2HPO4    
10 g yeast extract    
15 mg Thiamine add after sterilisation (filtered sterile)      
40 ml 25% Glucose add after sterilisation (filtered sterile)

All buffers and stock solutions should be prepared with diethylpyrocarbonate (DEPC)-treated H2O (DNAse/RNAse-free).
All stock-solution can be stored @ -20°C for weeks except creatine kinase. Creatine kinase has to be dissolved in 30 mM Glycin, pH = 9.0 + 20 mM DTT. After freezing in liquid nitrogen, it is stable @ -80°C for weeks.
All individual amino acids have to be dissolved as described on package. The mixture contains 5 mM of each amino acid with a pH of 7.5 adjusted with KOH.

Compound buffer
Tris/OAc, pH 8.2
KOAc
Mg(OAc)2
DTT (prepare fresh)
ß-ME (prepare fresh)
Vol. needed for 1 litre of culture
Buffer 1
10 mM
60 mM
14 mM
1 mM
7 mM
150 ml
Buffer 2
10 mM
60 mM
14 mM
1 mM
-
10 ml
Buffer 3
10 mM
60 mM
14 mM
-
-
1500 ml

Preincubation Buffer: 0.75 ml needed for 1 litre of culture

 
vol. of stock
stock
300 mM Tris Ac, pH 7.6 225 µl 1M
10 mM Mg(OAc)2 7.5 µl 1M
10 mM ATP 75 µl 100mM
80 mM phosphoenol pyruvate (PEP) 60 µl 1M
5 mM DTT 3.75 µl 1M
40 µM Amino Acid mix 6 µl 5 mM
8 U/ml pyruvate kinase 3 µl 2 U/µl
DEPC-H2O   ad 750 µl
  • Prepare an overnight preculture in 10 ml medium in a 100 ml Erlenmeyer-flask @ 37°C, 250 rpm, that will have to be inoculated in 1000 ml media in a 5000 ml chicanery-flask and grown till OD600 1.5-2 at 37°C, 150-180 rpm. Care about contaminations, because no selective-marker is added!
  • Centrifuge using SLA rotors at 6.000g, 30 min, 4°C, store pellet @ -80°C
  • All following steps should be done on ice!
  • Resuspend pellet in buffer 1 (16.6 ml buffer / gram pellet) by shaking or knocking by hand. Don´t pipet up and down!
  • Centrifuge @ 5.000g, 20min, 4°C and discard supernatant.
  • Resuspend pellet in buffer 1 (6.6 ml buffer / gram pellet) by shaking or knocking by hand. Don´t pipet up and down!
  • Centrifuge @ 5.000g, 20min, 4°C and discard supernatant.
  • Resuspend pellet in buffer 2 (1.3 ml buffer / gram pellet) by shaking or knocking by hand. Don´t pipet up and down!
  • Destruct cells using French press @ 6000 psi cell pressure (390 - 400 psi using the 1´´-piston @ high ratio) for only one time!
  • Pellet the lysate using ultracentrifugation @ 30.000g (around 20.000 rpm in Ti60 rotor) 30 min, 4°C (a large grey-green pellet will be formed). Transfer supernatant in new tubes and centrifuge again (a very small and clear pellet will be formed).
  • Transfer supernatant by using a pipette into 15 ml tubes and incubate in preincubation buffer (1 ml buffer / 10 ml lysate) @ 37°C, 90 min by using a roller (mid-speed) in the dark (wrapped in alu foil). The lysate will be cloudy.
  • Transfer lysate in high pure dialysis membrane (without heavy metals or glycerine, e.g. Roth V-Serie) mwco: 5.000 kDa and dialyse @ 4°C, 1h against buffer 3 (ratio 1 ml lysate to 100 ml buffer 3), change buffer and dialyse again @ 4°C, overnight.
  • Transfer lysate into 15 ml tubes and centrifuge @ 4.000g, 4°C, 10min.
  • Decant the supernatant and store in aliqoutes of 500 µl.
  • Shock freeze in liquid N2 and store @ -80°C.

Preparation of the S30-lysate with expressed T7 RNA polymerase (optional)

In order to have T7 RNAP in your final cell-free reaction, you can use a commercial T7 RNAP preparation or use T7 RNAP purified in your lab. Note that increasing amounts of glycerol might inhibit the cell-free reaction, therefore only highly concentrated T7 RNAP should be used (~1 KU/µl) in the cell-free reaction.

However, why use a protein purified from E. coli in order to add it to E. coli lysate? It is more convenient if you use a strain which can express the T7 RNAP gene upon, e.g. strains containing the DE3 lysogen with a copy of T7 RNAP like BL21(DE3) or a strain with a T7 RNAP expression vector. You can use the same protocol as above with a small modification:

  • Prepare an overnight preculture in 10 ml medium in a 100 ml Erlenmeyer-flask @ 37°C, 250 rpm, that will have to be inoculated in 1000 ml media in a 5000 ml chicanery-flask and grown till OD600 0.8 to 1 at 37°C, 150-180 rpm.
  • Induce the T7-RNAP production by adding 1 ml 0.5 M IPTG and grow till OD600 3 to 4

The subsequent steps are identical with the first protocol.

Note: You have to find an optimum ratio for the standard and T7 lysate in order to have highest productivity in the reaction mix (the T7 lysate is generally less productive than the standard lysate, thus you shouldn't use only the T7 lysate). A ratio of 1:5 or 1:10 (T7:standard) should be tested initially.

Reaction buffers

The final reaction mix is set up according to following table (note: there are varying concentrations for some of the components in different publications, we have listed the ones that we found to work best in our hands, you can try other variants as well)

Energy Mix (3.2x):

(Can be stored at -20° for several months)

 
stock
 
final conc.
 
Vol. [µl]
DTT
1000
mM
1,7
mM
19,7
NTPs *
30
mM
1,2
mM
464,0
cAMP
100
mM
0,65
mM
75,4
Creatine phosphate (CP)
3000
mM
80
mM
309,3
tRNA (E. coli)
87,5
mg/ml
0,175
g/l
23,2
K-Glutamate
4000
mM
200
mM
580,0
PEG 8000
30
%
2
%
773,3
EM Buffer
10x
     
1160,0
ddH2O        
220,0
Total        
3625,0

* NTP stock solution contains 30 mM ATP and 20 mM each CTP, GTP und UTP (final concentration of GTP/CTP/UTP of 0.8 mM).

10 x EM buffer:
(for 10 ml final volume)

 
stock
 
final conc.
  Vol. [µl]
µl
HEPES/KOH, pH7.5
1380
mM
550
mM
3985,5
µl
Folinic acid
1
Mg/ml
350
µg/ml
3500,0
µl
NH4OAc
5000
mM
280
mM
560,0
µl
Mg(OAc)2
1000
mM
110
mM
1100,0
mg
NaN3    
0,5
%
50,0
µl
ddH2O         854,5
µl
Total         10000,0  

Setting up a cell-free reaction

Depending on the protocol you are using there are variations in the reaction composition and concentration of the components. This indicates that the system is quite robust and further optimization of the cell-free reaction mix is possible.

Final Reaction Mix:

Component
V [µl]
Lysate
400,0
Lysate T7
50,0
Energy Mix (EM, 3.2x)
312,5
Amino Acid Mix ** (2mM final each)
80,0
Creatine kinase (80µg/ml final) ***
4,0
Vector DNA (10µg/ml final)$
x
Additives (e.g. detergents, cofactors, ions, etc)
0,0
ddH2O
153,5-x
Total
1000,0

** Note that the Amino Acid Mix has to be 25mM for each of the 20 AAs, some of them will not be soluble at pH 7.5-8 which should be the pH value of this mix. It is usually no problem to add it as a suspension after thorough mixing.
*** Creatine kinase should be dissolved at 20mg/ml in 30 mM Glycin, pH9.0 + 20 mM DTT and stored in small aliquots at -80°C. Avoid multiple freezing and thawing.
$ Vector DNA should be of high quality (Maxi-prep)

In case of further questions, please refer to following references or send us your comments or questions to pepcore@embl.de.

References

  • Zubay (1973), "In vitro synthesis of proteins in microbial systems", Ann. Rev. Gen., 7, 267-87
  • Kigawa et al. (1999), "Cell-free production and stable-isotope labeling of milligram quantities of proteins", FEBS Letters 442, 15-19
  • Kigawa et al. (2004), "Preparation of Escherichia coli cell extract for highly productive cell-free protein expression" J. of Struc. & Funct. Gen. 5, 63-68
  • Kim et al. (2006), "Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system" J. Biotechnol., 124(2), 373-80

Necessary components

We recommend using chemicals of high quality, do not always go for the cheapest one on the market. Once established, you can test if replacing single components with cheaper ones is feasible and, above all, really necessary. Remember, that most components will be purchased in large amount and last for a very long time (e.g. amino acids), it doesn't make sense to save money there in order to have a final saving of a couple of cents in the final reaction.

E. coli strains:
A19, BL21 CodonPlus, etc.

HEPES-KOH, pH 7.5
KOAc
K-glutamate
Tris-acetate, pH8.2
Mg(OAc)2
NH4OAc
PEG8000
DTT
ß-ME
NaN3

20 amino acids
NTPs (ATP, GTP, CTP, UTP)
3',5'-cyclic AMP (cAMP)
Folinic acid

phosphoenol pyruvate (PEP)
pyruvate kinase
creatine phosphate
creatine kinase
T7 RNA polymerase
total E. coli tRNA