ProteoCool Pills#14: Hints for cell propagation and recombinant protein expression using Expi-CHO and Expi293 (WT and GNTI-)

For many years the application of the traditional adherent mammalian cell cultures for recombinant protein production was difficult and applicable only in those approaches requiring very low protein amount (ug’s) because cell scale up was very difficult, transfection not very efficient and the final volumetric yields (mg/litre) very low. 

This limitation forces the scientist to use other simple eukaryotes expression systems as Pichia Pastoris or Baculo virus for the production of mammalian protein in mg scale.

However, in the last 20 years some technological improvements as:

 1) The adaptation of several cell lines to suspension cultures;

 2) The development of more efficient transfection agents;

 3) The development of serum free media where the cells are able to growth at high cell density;

Improve drastically the performances of the mammalian expression systems;

As I already mention in previous presentations, theExpi293 and Expi-CHO date, represent 2 of the most performing mammalian transient expression systems. 

If it is true that those systems are quite expensive, on the other hand they are very simple to use and guarantee, high yields also in shake flask. 

For some recombinant protein, Expi293 in shake flask were able to provide me up 300mg/yields. 

Values that until some years ago were reachable only using E. coli expression in high cell density set-up (e.g. feed-batch fermenters)

Expi-CHO are able to provide up to 100-200 mg/l of full length recombinant antibodies. Full length antibodies are big (150KDa), composed from 2 chains and contains several disulfide bonds, their expression in bacterial expression system is very challenging.

However, to reach those results details, as;

-             - The selection of the optimal shake flask format and right culture volume;

-              -  The selection of an antibiotic not affecting cell growth and protein expression;

-                The selection of the optimal signal peptide for protein secretion into the cell surnatant;

can make a huge difference!!

The selection of the right shake flask and culture volume

Expi293F and Expi293 GNT- cells :

In small scale (up to 60ml culture) Expi293 are not so sensitive to the flask format and presence of baffle:

For example, in our experience Expi293 are growing well in both

-        Erlenmeyer flaskscup vented plain (cost ~ 8€/flask for 125ml format)

-        Erlenmeyer flasks cup vented baffled

-        Thomson Optimun growth flaks  (cost ~ 8€/flask for 125ml format and 30€/flask for 1,6l)

Optimal culture volumes: 

125ml flask volume for 30ml Expi293 transfection and 250ml flask volume for 60ml Expi293 transfection

For culture scale up (eg 200-500ml culture),  the Thomson Optimun growth flaks eems to perform better probably  because it guarantee better CO2 and oxygen exchange.

My preferred format is 200-300ml transfection in 1.6liter flask and generally we do not exceed the 500ml single transfection (using a 2.4 litre flask) because in high volumes we more frequent bacterial contamination was observed, therefore in case of large culture volume e.g. 1-2liter, I prefer to split the culture using more flask in parallel.

ExpiCHO:

ExpiCHO show an high tendency to aggregate and are much more susceptible to  the flask format.

Thomson Optimun growth flaks https://htslabs.com/og/ that contain small baffles seem to represent the best compromise and are the format more able to prevent the cell aggregation during the cell maintenance passages.

N.B: Expi-CHO are able to growth up to very high cell density (>10milion/ml) but to avoid cell aggregation is it better do not exceed the 6 milion/ml during the cell propagation passages

Optimal culture volumes: 

   125ml flask for 25ml transfection 

   250ml flask for 50 ml trasfection

    1,8liter flask for 150-300ml tranfection

In all cases we use the max titer protocol à 8day of trasfection at 32°C

The selection of the right antibiotics to prevent cell contamination.

The Expi293 and ExpiCHO Thermofisher manuals do not suggest the addition of any antibiotics to prevent bacterial contaminations. However after that we experienced some culture contaminations, considering the high  cost of the reagents (more than 1000euro/Liter)  we decided to perform some  transfection trials in the presence of  antibiotics to see if they affect or not the cell viability, growth rate and recombinant protein expression

Expi293 are not affected by:

-        Pen/strep (thermo cod. 15070-063) diluted 1:100 (50 unit/ml penicillin and 50ug/ml Streptomycin)  

-        Gentamicin (Sigma cod. G1272) diluted 1:200 (50ug/ml)

Generally, we use for cell propagation and transfection the Pen/Strep 

Expi293 GNT- are not affected by Gentamicin (Sigma cod. G1272) diluted 1:200 (50ug/ml) while reduction in cell growth rate and viability was observed in presence of Pen/Strep;

Expi-CHO are not affected by Pen/strep (thermo cod. 15070-063) diluted 1:100 while reduction in cell growth rate and viability was observed in presence of Gentamicin diluted 200 times.

 

 

 

 

 

ProteoCool Pills#13: Densitometric Protein quantification from SDS-page using the Image J free software package

 Several different methods are currently available to perform quantification of purified recombinant proteins and antibodies

There is not a best, universal method, the provide a reliable result for all the proteins;  

Each methods have it some prons and cons and its applicability depend from the intrinsic properties of the target protein.

For example:

UV quantification that exploits the properties of aromatic amino acids (tryptophan and tyrosines) to absorb energy around 280 nm is fast and require limited amount of sample but cannot be performed with proteins that do not contain aromatic amino acids or with buffers with an intrinsic absorbance in the UV regions.

Colorimetric-fluorimetric assays as Bradford, BCA, Nanoorange are susceptible to buffer compositions (eg BCA is not compatible with reducing agents) and to extrapolate quantitative results, the comparison with a  calibration line is required Results may change a lot on the basis of the protein that is used to build the calibration line (generally BSA) because different proteins may show different response in function of their aminoacidic composition or stability of their conformation in presence of the dye.

gg: Bradford assay is less sensitive to full length antibodies (igG) than BSA (see fig 2 page 6 ) and therefore in case you would like to use Bradford assay to quantify a monoclonal antibody (mab) a calibration with a commercial mab is required.

In some unlucky cases, for those proteins that do not contain hydrophobic amino acid and shows low response to colorimetric assay (due to strong conformational stability of presence of post translational modifications, eg hyper glycosylation) all the previous methods may not be reliable and densitometric analysis from SDS-page may represent a simple and cheap alternative.

Quantitative densitometry of proteins from SDS-page stained with colorimetric reagents (eg  coomassie blue) require a software to perform image processing,  extrapolate peak area and correlate it with the sample concentration.

To date most of the commercial gel documentation systems are supplied with their Image analysis Software able to perform band intensity determination.

However, if those Gel acquisition systems are still essential for acquisition of agarose gel images, high quality images of SDS-page gels stained with Coomassie can be obtained using modern smartphone those carrying high resolution camera.

ImageJ (NIH), a public domain program from the National Institutes of Health downloadable at https://imagej.nih.gov/ij/download.html can be used to analyse the SDS-page images.

1. Open the gel image

On the gel selected for this example, we load several dilutions of a purified protein sample with unknown concentration (to be determined) and several know amount of BSA required to build a reference calibration curve

2. Select rectangle in the AREA SELECTION TOOL

3. Choose the 1^st  line, select the rectangle tool, and draw a box around the lane, 

making sure to include some of the empty gel between lanes and white space outside of the band. 

When creating the selection, drag with the shift key down to constrain it to a square.

4. Define the 1^st  line: Go to Analyze→Gels→Select first lane

      5. Select the 2^nd line

        Make sure your cursor shows as an arrow, grab the rectangle you just made, and drag it to the next lan

DO NOT DRAW NEW RECTANGLES! You must drag the same rectangle you just made because to compare the band you have to use the exact same size originally defined area in Lane 1.

6. Define the 2^nd line: Go to Analyze→Gels→Select next lane

 

7.  Repeat the step 5 and 6 since all the line (sample and standard dilutions) are selected and numbered

 8. Go to Analyze→Gels→Plot lanes 

 

A new windows containing an histograms for each line will appear

9. Drag two fingers on the mousepad to scroll up and down and navigate the grids

The peaks in each grid correspond to the intensity of the bands in the lane

 10.   On the ImageJ interface, select the "line" button (red arrow) to define the peak baseline

     11. Draw a line at the bottom of the peak that represents the baseline of your peak and it allow to define the area of the curve.

12. Drag two fingers on the mousepad to scroll down and keep drawing all the single lines to define the curves in your standard and protein of interest lanes.

 

  13. Once you draw a baseline for each peak, on the ImageJ interface, select the "magic wand" button (red arrow)

 

 14. Click on the line defining the area of the curve of the first peak

 A "Results" window containing the measured area will appear

  15. Drag two fingers on the mousepad to scroll down and define the area of all peaks with the defined baseline

16. In the Result window Go to File→Save as

ans Save your Results in .csv format so that you can transfer the measurements to excel to generate the standard curve (linear regression analysis) and determine the concentration of your protein sample.

A Possible mistakes:

When you draw the peak baselines (point 10-12), the line has to interpolate both “feet” of each peak 

to correctly define and measure the peak area. 

The same analysis could be used to determine band intensity and extrapolate dna or rna quantification from agarose gel. 

However in my opinion the limited linear range of densitometric analysis and low reproducibility in gel load and coloration make this quantification approach not very precise and have to be applied only when better alternatives (as 280nm quantification for DNA) are not available.

ProteoCool Pills#12: Rapid insertion/deletion/replacement of N-terminal signal peptide sequences using PIPE cloning

 In both eukaryotic and prokaryotic cells, all proteins are synthesized in cytoplasm. 

Proteins that are destined to enter into the secretory pathway are usually endowed with an N-terminal signal peptides (SPs, known also as N terminal leader sequences): the signal peptide those are short peptides and usually have a length of 16–30 amino acids.

After directing proteins to their specific locations, SPs are removed by signal peptidases

The presence or absence of the SPs allow to direct the expression of the protein in different cellular compartments:

- E.coli,  a protein with-out SPs will be directed in the cytoplasm, while the addiction of a SPs (as pelB, OmpA signal peptides) of the signal will  direct the protein into the periplasm;

-In Gram positive bacteria (as baccilus) and mammalian cells (as HEK293, CHO) the addiction of N terminal SPs direct the protein in the culture surnatant.

Since the accumulation of recombinant proteins in the cytoplasm may lead to the formation of inclusion bodies or protein degradation via proteases and the recombinant protein folding may also affected from the reducing conditions of the cell compartment (eg E.coli cytoplasm is strongly reducing and not compatible with S-S bond formation), the selection of the right Signal peptides play a decisive role in the industrial production of recombinant proteins.

It has been shown that using different homologous or heterologous signal peptides can affect the yields of recombinant proteins. Selecting a proper signal peptide to increase the secretion efficiency becomes a common methodology to optimize the production of recombinant protein

The availability of a simple cloning method to readily add, replace and modify a signal peptide sequences in an expression clone is therefore an essential tool to screen for the best protein/antibody productivity.

As already mentioned in the ProteoCool n°1: Cloning methods overview; The PIPE cloning is a nice method to manipulate expression vectors and perform mutagenesis, insertion and/or deletion or some vector regions.

With a single vector PCR is possible to insert dna fragments up to 80-100bp in any  vector region and therefore it can be applied also to the insertion of replacement of signal peptides in the vector of interest.

Using the PIPE cloning i  was able to insert pelB (MKYLLPTAAAGLLLLAAQPAMA)  and ompA (MKKTAIAIAVALAGFATVAQA) signal peptides in several pet15 clones for E.coli expression and other mammalian signal peptide in pcdna 3.4 clones for expression in Expi293 and Expi-CHO.

Of couse a similar approach could be performed to insert/delete or replace other short sequences as:

 - 6x His Tag

- Avi Tag

- Protease digestion sites (eg TEV, Eterokinase, Fatt.Xa)

or delete:

-N- or C- teminal region of your gene;

-N or C- terminal fusions (eg GFP, GST, MBP, etc)

----------------------------------------------------------------------------------------------------------------------------

PIPE cloning steps:

1) Vector amplification by PCR  (V-PCR)

2) PCR digestion with dpnI (to remove template vector background)

3) Trasfrom the vector into the MACH1 E.coli cells

4) Plasmid extraction from at least 4 colonies and DNA sequencing

-----------------------------------------------------------------------------------------------------------

                                            Tips to perform the Vector PCR:

PCR reaction:

My preferred DNA polymerases: Kapa Hifi (Roche) or Clone amp (Takara)

                       Theoretically you can use all high fidelity polimerases that do not add poly AA

DNA template --> less than 0.1ng/reaction 

(higher template amount may result in background colonies with the original template)

PCR volume --> 25ul/reaction are more than enough considering that for PIPE reaction 1-2ul are normally enough and PCR purification is not required.

PCR cycle:

Elongation time --> >1minute/kb also if the datasheet of the Taq suggest shorter extension time to exploit the 3-' --> 5' exonuclease activity that improve the formation of incomplete extension that are require for PIPE cloning.

Do not insert the final elongation time step.

---------------------------------------------------------------------------------------------------------------------------  

V-PCR trasfromation in E.coli cells (PIPE reaction)

PIPE cloning do not require PCR purification.

1)  Mix 2 ul of the V-PCR in 20ul of chemically competent MACH1 cells (thermo cod. )and incubate the mixture in ice 30 minutes;

N.B:  If the V-PCR band intensity is very high  (as in the reported picture)  yon can try also to dilute the plasmid 5 times and perform a second trasfromation with 2ul of the diluted plasmid in 20ul of cells because too much DNA can reduce the trasformation efficiency)

2) Incubate the cells 1' at 42°C

3) Transfer the cell in ice

4) Add 250ul of SOC or LB sterile media (with out antibiotic)

5) incubate the cells at 37°C - 180/600rpm (in a thermomixer or incubator shaker) 

6) Plate all the cells in LB-agar plates containing the proper selection antibiotic

(eg 100mg/l ampicillin for pet21 or pcdna 3.4,  50mg/l of kanamicin for pet24 clones)

7) incubate the plates O/N at 37°C

Primers design

Annealing regions à 18-26bp with an annealing temperature salt adjusted (calculated with Oligocalculator)  of 58-62°C if is possible)

Flanking regions –> length up to 60bp. Overlapping regions have to be between 15 and 20bp

Is not mandatory to add flanking regions in both forward and reverse primer to create the 16bp overlapping. It can be done also adding the flanking region in just 1 of the 2 primers. I choose one or the other solution on the basis of the length and Tm of the relative annealing regions.

----------------------------------------------------------------------------------------------------------------------------------------------------                                                                                      A great thanks to  

Roberto Petracca

an amazing supervisor who introduced me to the PIPE cloning

i was very lucky to shave with him many years in the Novartis Reseach centre in Siena 

References: 

Klock HE, Lesley SA. The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis. Methods Mol Biol. 2009;498:91-103.