ProteoCool Pills#11: Chemical_enzymatic cell lysis as alternative to physical lysis in high-throughput small scale protein purification from E.coli

 

Different methods can be used to perform Cell lysis for protein isolation and purification from Escherichia coli (E. coli) cells including sonication, homogenization, enzymatic lysis chemical lysis and freezing/grinding.

Ideally the perfect method has to be cheap simple, scalable, efficient but also gentle to allow complete cell lysis but non-denaturing to preserve protein structure and function.

Unfortunately, in the real world, a best universal method does not exist:  each method has its pros and cons that make it preferable on the basis of the amount of the cells and the number of samples to be processed in parallel.

-The use of Freeze/Thaw (coupled or not with enzymatic lysis; eg Lysozyme supplementation) is preferable only when we have to manage an high number of small samples, the efficiency of this lysis approach decrease with increase in biomass:

- The sonication is the most used approach in lab scale to produce mgs of recombinant protein.  Sonication can be used with biomasses from mg to 10-100g with a limited number of samples. With small biomasses up to 24 sample in parallel can be processed using a 24- probe sonicator – See Tips ofthe week #6)

The sonication cycle needs to be carefully optimized on the basis of the sonicator power, sonicator probe and sample volumes to obtain complete cell lysis but avoid localized heating within a sample that may induce protein unfolding and aggregation

 -   High pressure Homogenizers are the most commonly used devices used in industrial scale to lyse large quantities of bacteria but they do not allow to manage multiple samples in parallel.

- Combination of Chemical/enzymatic cell lysis could be an interesting option, alternative to the sonication, for all those projects (e.g protein production for microarray or preliminary crystallization screening with nanodrops) that require to produce in parallel limited amounts (0,5-1mg) of many different proteins.

Chemical lysis is generally based on the use of moderate concentration of anionic or zwitterionic detergents which are mild and therefore able to compromise the integrity of cell membranes and facilitating lysis of cells and extraction of soluble protein maintain their native form.

In the past I had the opportunity to use 2 different commercially products suitable for Chemical/enzymatic cell lysis:

CelLytic™Express (Sigma Aldrich cod. C1990 ) and B-PER in phosphate buffer (Thermo cod. 78266 )

CelLytic™ Express is a non-denaturing formulation including a zwitterionic detergent (CHAPS:  3,7-5% as reported in the formulation sheet), lysozyme and endonuclease applicable to protein extraction from E.coli before affinity purification (e.g. IMAC)

B-PER a non-denaturing formulation including a non-ionic detergent (unknown; non reported in the certificate of analysis provided in the Thermo website) in phosphate buffer. Prior use it has to be supplemented with lysozyme. DNase and MgCl2.

In some rare cases (as the following) the Chemical_enzymatic Lysis was able to improve the protein solubility respect than sonication

 

In my experience the performances of CelLytic and B-PER are similar and which perform better is not predictable but there are differences protein to protein, however If i have to choose one of those, my first choice is the CelLytic which seem to have slightly more frequently better impact on protein solubility.

                                                                    My personal choice:

 A) If you can buy a good sonicator (eg Q700 Qsonica ) carryng a 24 tips probe:

 Sonication: 1^st choice while Cell lytic 2^nd choice to be tested as recovery plan of those proteins that result not soluble with sonication.  

 B) If you do not have a multiprobe sonicator:  CelLytic may represent a good 1^st choice when you have to express and purify more than 4 protein sample in parallel.

N.B: Whatever method do you like, remind to use the same method to evaluate protein solubility in the small scale expression scale as well as you perform the scale-up protein purification.

DON’T use Chemical_enzymatic Lysis in small scale to screen solubility of a pellet just to save time and then perform the lysis of the entire biomass with sonication or high-pressure homogenizers because in some cases the results are not corresponding.

Pros of CelLytic

- Simple and fast protocol:

1) Resuspend the CelLYtic powder of a bottle in 25ml of water

2) Resuspend the E. coli pellet adding 10ml of Celllytic/g E.coli pellet

3) Incubate 30 minutes at RT under agitation. (Take sample for SDS page –> total fraction)

4) Centrifuge and filter surnatantthe  at 0.22um

5) Perform affinity purification (eg.IMAC)

- Do not require expensive instrumentation (eg Sonicator)

- Compatible with affinity purification (eh IMAC);

Cons of CelLytic

- Reagent is Expensive à  250ml of Cellytic cost about 230euro à 250ml for 25g of wet biomass

                                              About 10 euro/gramm biomass!

- Sample is not directly appliable in SDS-page, because the presence of the detergent affects the sample run. The sample need to be diluted at least 4 time in a buffer (e.g. IMAC binding buffer) with-out detergents.

-

A great THANKS to

Roberta Cozzi that introdued me to the use of CelLytic express and

Roberto Petracca that introduced me to the use of B-PER

!! Work with both of them was a great pleasure !!

!! I miss a lot the 2 Ro’!!

ProteoCool Pills#6: Rapid protein Expression and solubility check using a 24 tip-horn sonicator

 

The use of high cell density media (e.g Enpresso reported in the ProteoCool n°7 or YGR reported in the ProteoCool n°32 ) for protein expression using E.coli allows to scale down culture volumes and easily express many clones in parallel.

Protein expression and solubility check can become a limiting step and the set-up of a simple and reliable protocol is essential.

Automation by using robotic platform is a possible solution for a real high-throughput approach, (e.g a screening projects that require to process  thousands samples for week) but in the most cases just the implementation of a optimized and rational protocol based on the use of multi-channel pipettes and 96-deep well plates can allow to manipulate in parallel up to hundreds samples/week with low cost and high protocol flexibility.

The lysis method used in the small scale test need to be similar to the ones that will be applied for the purification of the protein from rest of biomass in case of positive results, and therefore Sonication or is preferable respect than freeze-thaw cycles (work well in small scale but is not scalable) and chemical/enzymatic lysis ( expensive in scale-up)

Protocool

1) About 8OD of bacterial cells (for Enpresso correspond to 800ul not induced or 500ul induced 8h or 300 ul induced 24h) were transferred from culture into the 96 Deepwell plate following the scheme reported below (to fit the 24 tip sonicator horm)

2) Plate were centrifuged for 20' at 3700 rpm and 4°C

3) After centrifugation the surnatants were discharged 

4)    The pellets were resuspended adding 960ul of cell Lysis/binding buffer ( eg: Tris 20mM, NaCl 300mM, imidazole 10mM pH=8 for His-tagged protein that will be purified by IMAC) and mixed 3-5 times with the multichannel to allow complete pellet re-suspension. 

2)    Cell Lysis by sonication using the 24-tip horn:

Sonication cicle: 30’’ ON/45’’ OFF , 20 cycles  50% power – Qsonica Q700 sonicator.

The plate need be placed in ICE during the sonication to avoid sample heating.

3)    From each well 60ul of sonicated solution (Total fraction) e transferred in PCR plate no skirted containing 20ul of Loading sample buffer 4X ( eg. Thermo scientific cod. NP0008 ) with reducing agent.

4)    The 96-deepwell plate were centrifuged at 20’ a 4000rpm 4°C;

5)    From each well 60ul of surnatant (Souble fraction) e transferred in PCR plate no skirted containing 20ul of Loading sample buffer 4X ( eg. Thermo scientific cod. NP0008 ) with reducing agent.

6)    The no skirted PCR plate is closed by aluminum foil and incubated 5’ a 95°C in a PCR machine.

7)   12ul of each samples were loaded in a 26well 4-12% SDS-page gel.

Example of results: