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#10: Use of 2F-Peracetyl-Fucose to produce low fucosilated monoclonal antibodies with Expi293 and ExpiCHO cell lines

Therapeutic monoclonal antibodies are the fastest growing class of therapeutics for the treatment of various cancers and inflammatory disorders. The Fc region of human IgG antibodies interacts with multiple Fcγ receptor (FcγR) and complement proteins and mediates immune effector functions, which are important for many therapeutic applications, e.g. elimination of targeted cells via antibody-dependent cellular-cytotoxicity (ADCC), -phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC).

To date, therapeutic IgG antibodies (either approved or in clinical development) belong to the IgG1, IgG2 or IgG4 subclasses. Each IgG isotype has a distinct binding affinity to the various FcγRs, which are expressed differently on immune cells. 

In cancer immunotherapy, some IgG1 antibodies (eg ipilimumab) rely on the Fc-mediated immune effector function, dependent cellular cytotoxicity (ADCC), as the major mode of action to deplete tumor cells.

It is well-known that this effector function is modulated by the N-linked glycosylation (N297) in the Fc region of the antibody.

In fact, if the N297A mutation  is able to reduced FcγR-binding and abolish ADCC activity on the other hand,  the absence of core fucose on the Fc N-glycan has been shown to increase IgG1 Fc binding affinity to the FcγRIIIa present on immune effector cells such as natural killer cells and lead to enhanced ADCC activity.

Various strategies have focused on producing afucosylated antibodies to improve therapeutic efficacy. While engineering of the cell lines (eg FUT8 deletion: @Potelligent or @GlymaxX Technologies) are required for scale up production of recombinant afucosilated mabs, for small lab scale for production of mgs, the supplementation of 2F-Peracetyl-Fucose is a simple and efficient solution to produce “low fucosilated mabs” to be used in functional in vivo and in vitro assay.

Media supplementation with 200 µM of  2F-Peracetyl-Fucose allow to produce low fucosilated mabs with transient transfection in Expi293 or ExpiCHO cells) just before cell transfection (as reported on Chakraborty et.al for the Expi293 cells) allow to produce a mab with low fucosilation level (as reported in Fig1) and enhanced ADCC activity.

n.b: The results of ELISA assay on mabs produced from Expi293 and ExpiCHO seem to suggest that the ExpiCHO show a decreased fucosilation level respect than Expi293 also in absence of the 2F paracetyl fucose. 

Preparation of 2F-Peracetyl-Fucose stock solution:

2F-Peracetyl-Fucose is supplied as powder (10mg). It can be directly resuspended in the glass bottle with 1mM DMSO to obtain a 34.2mM stock solution (171X) that could be stored at -20°C.

584µl of 34.2mM of 2F-Peracetyl-Fucose were added to 100 ml of cells just before the transient transfection with Expifectamines

Prons:

Very Simple!!

Concerns:

Expensive: Cost of  2F-Peracetyl-Fucose: ~ 1000euro/liter of transfection make this approach feasible only for small lab scale productions

ProteoCool Pills#9: Use of low purity imidazole in buffer for IMAC may prevent protein detection during elution steps

Although imidazole itself should not give a UV280 signal, many commercially available products contain impurities which can give a strong UV signal.

Eg:   Absorbance spectrum of low purity imidazole buffered solution vs (black line) vs high purity imidazole (red line) acquired with nanodrop

The high imidazole concentration present in the IMAC Elution buffers (250-500mM) therefore the use of an imidazole with high Absorbance at 280nm may determine the detection of a plateau of absorbance during the elution (eg. Fig A) which may cover partially or completely the peak of eluted proteinFig.A:  His-Tagged protein purified from Expi293 surnantant with IMAC chromatography using an Elution buffer containing a low purity imidazole. 

                                                                    Solution (see Fig.B)

Be sure to use highly pure imidazole (eg Merck cod. 1.04716 ) which gives essentially no absorbance at 280 nm

Fig.B:  His-Tagged protein purified from Expi293 surnantant with IMAC chromatography using an Elution buffer containing an high purity imidazole. 

Acknowledgments:

" I would say a great thanks to Mirco Toccafondi, a great biochemist ever open to share his knowledge with the others !! "

 

ProteoCool Pills#8: Expi-CHO a powerful cell line for transient expression of biologics

 

If in the ProteoCool n°29 I extensivelly show the properties of the mammalian Expi293  expression system, today I would like to briefly report you the further improvement in terms of productivity that I was able to obtain thanks to the ExpiCHO cell expression system.

Example 1: Expression of a mouse igG2a-CL-k recombinant monoclonal antibodies 

 (both heavy and light chain were cloned in pcdna3.4) with 2 different leader peptides)

 

Protocol (in brief):

1)     15 ml of cells were transfected in a 125 cap vented flask

2)     Cultures timelines:

1)     Cell were centrifuged 20’ at 3000g and surnatant was filtered at 0,22uM

2)     Surnatant is added of 1.5ml of PBS 10X

3)     Surnantat is loaded on a Poliprep gravity column containing 300ul of Protein G FF resin

 5b) For the Expi-CHO productions, the Flow-Through from the column was loaded in a 2nd protein column to recover all the mab

4)     Column is washed with 50CV of PBS

5)     Mab was eluted with glycine 100mM ph2.7 on a Tris 1M pH=9 solution

6)     For Expi-CHO cells due to high expression the 

7)     Mab was concentrated with amicon ultra 50Kda and buffer exchanged in PBS

8)     Mab was quantified with nanodrop measuring the 280nm absorbance and the igG quantification mode.

Results:

                      !! ExpiCHO vs EXpi293 --> More than 10 fold improvement!!

Example 2:  Production of recombinant  human CTLA-4 (1-161 domain) 

(carrying a C-terminal 6XHis tag cloned in pcdna 3.4)

Protocol (in brief)

1)     15 ml of cells were transfected in a 125 cap vented flask

2)     Cultures timelines: Same that reported in the Example

3)     Cell were centrifuged 20’ at 3000g and surnatant was filtered at 0,22uM

4)     Surnatant is added of the same volume of IMAC binding buffer (Tris 20mM, imidazole 10mM, NaCL300mM ph=8)

5)     Surnantat is loaded on a Poliprep gravity column containing 300ul of Ni-sepharose FF

6)     Column is washed with 50CV of Binding buffer

7)     CTLA-4 was eluted with 1ml of IMAC elute buffer (Tris 20mM, imidazole 300mM, NaCl 300mM pH=8.

8)    CTLA-4 was quantified with nanodrop measuring the 280nm absorbance. CTLA-4 extinction coefficient was calculated by ProtParamTool

 Results:

Summary:

 

Pro/Cons Expi-CHO vs Expi293

 

Low DNA amount required for transfection (0,5ug/ml vs 1ug/ml)

Low temperature during protein expression(may help production of thermostable proteins)

Use in the preclinical studies the same cell line (eg CHO) that will be used for the manufacturing of clinical lot and that represent the gold standard strain for biomanufacturing and reduce risks associated to strain transition 

High growth rate at 37°C and higher tendency to aggregate: Routine cells passages require more attention to perform the cell count and cell dilution.

Longer culture times (at least 8day vs 4day)

PMT (eg glycosylation profile) may be slightly different from the human cells (eg HEK293 or Expi293)

2 different CO2 incubator shaker are required: shakr n°1 fixed at 37°C 120rpm 8%CO2 for  the cell line propagation a shaker n°2 fixed at 32°C 120rpm 5%CO2 for cell transfection

Expensive or cheaper? 

It depends from the yields. In the reported examples, much cheaper! 

To date culture media and transfection reagents are slightly similar

 

1liter Expi293 mediumà 340euro vs 1liter ExpiCHO medium à 466euro

1liter transfection kit Expi293 à1128 euro vs 1liter transfection kit Expi-CHO à1236 euro

  

Those costs may seem very high (about 2000euro/liter) but it depends from yields and complexity of each target.  

 

Expi293 and ExpiCHO allow to you to produce high yields of proteins (eg protein rich of S-S bonds) and/or  full length antibodies those are almost impossible to be produced in similar yields (and correct folding) using less expensive organisms (as E.coli)