Protein Expression and Purification Protocol

From Plasmid to Protein

Step 1: Transform appropriate DNA plasmid into BL21(DE3) E. coli cells. These cells must be competent. (Protocol for how to make competent cells.)

a) Take competent cell stock aliquot (about 100 mL) out of -80oC freezer. Place on ice to thaw about 5 minutes.

b) Take one mL of plasmid DNA (usually a pRSET B vector with your protein cloned inside DNA seq of pRSET B) and add to the thawed competent cells. Vortex briefly to mix, and let incubate on ice for at least 20 minutes. Longer incubation will not hurt.

c) Heat shock the competent cells at exactly 42oC for 1 minute. Place on ice.

d) Add heat shocked cells to 1 mL of LB broth in a sterile culture tube, but DO NOT ADD antibiotic. Shake at 37oC for at least 1 hour. Shaking for 3 hours doesn't seem to hurt.

e) Plate the cell culture onto LB agar plates (usually 100 mL of culture will do although it depends on the competency of the cells and the initial concentration of plasmid). Make sure the plates have the appropriate antibiotic (usually ampicillin at 100 mg/ml concentration). (Protocol for how to make LB Ampicillin Agar Plates) Incubate the plate at 37oC for 14 hours (overnight generally). Longer times of incubations will allow for satellite colonies to appear.

Step 2: Make a starter culture for protein expression. Usually to about 250 to 500 mL of LB broth the antibiotic is added.

a) Generally, the antibiotic is ampicillin, which is added to the LB broth at 100 mg/ml final concentration. (Have a stock of ampicillin in the 4oC which is 1000x concentrated or 100 mg/ml in sterile distilled water to make things easier).

b) Pick a colony or two from your plate using a sterile innoculation loop and add to the LB broth/ampicillin flask. Incubate in a shaker (250 RPM) at 37oC until the OD @ 600 nm is 0.5 to 1.5 OD. (Anywhere in this range is fine, however, extremely dense starter cultures>1.5 OD can cause problems in protein expression. Under these dense growth conditions the plasmid seems to be rejected or turned-off by BL21(DE3)s.)

c) Save your starter culture flask by placing in the 4oC overnight. This will slow down the bacterial growth enough, but prevent the problems dense cultures cause as listed above.

Step 3: Make the big batch of bacteria culture for protein expression. (How many 1.5 L volumes should you grow up? Expect about 100 mgs of protein per liter, but that estimate is very dependent on the protein!)

a) Equally divide your starter culture among several 1.5 L volumes of sterile LB broth in 2.8 L Fernbach culture flasks. (Good precision when equally dividing the starter will better control the timing when induction can occur.)

b) Add antibiotic to 100 mg/ml final concentration. Grow them up in a New Brunswick shaker large enough to hold several Fernbach flasks.

c) Grow until an OD 600 nm of 0.8 to 1.2. Then induce the culture to express protein by adding 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. This is expensive stuff so use it carefully. Only measure out exactly the amount of IPTG you need for your flasks. Dissolve that amount in about 10 ml of sterile water and divide it equally among your 1.5 L flasks. This stage is called induction. Keep the culture shaking at 37oC.

d) Induction of protein generally takes 3 to 4 hours (but this too depends on your protein). After induction, centrifuge your bacteria in 500 ml bottles in the big Sorvall rotor at 5,000 RPM (bigger than the GSA found on the 2nd floor with Rice, Correl, and Moffat Labs). Do this in batches until all your culture is spun down saving the cell pastes each time. Freeze the cell paste at -80oC.

Step 4: Lysis and sonication of the bacteria. There are many ways to lyse bacteria, skin chickens etc., but this method is tried and true.

a) Make Lysis Buffer: 25 mM TRIS-Cl, 2 mM EDTA, pH 7.6. (You may add protease inhibitors like benzamidine or PMSF if you like but add these right before you start to lyse. Make concentrated PMSF solutions in pure ethanol since it is hydrophobic.) Then add lysozyme at 100 mg/ml concentration (or just a tipful from a spatula). Generally the volume lysis buffer is 1/20 to 1/50 the volume of the bacterial culture.

b) Resuspend the frozen cell paste as best you can in the Lysis Buffer using a 10 ml pipet or whatever means necessary. Let this suspension incubate for 20 minutes at room temperature, or until the suspension becomes turbid and viscous due to release of the bacteria's genomic DNA.

c) In order to eliminate the extreme turbidity of the suspension, sonicate the suspension to shear the DNA until the turbidity is similar to that of a normal protein solution. Then centrifuge at 18,000 RPM in a Sorvall SS-34 rotor (or ~40,000 x g) for 20 minutes at 4oC.

d) Save the pellet and the supernatent. (If the solution is slighly turbid due to residual DNA, a quick way to shear the DNA is to pass through a syringe with a needle.) Generally your protein is in the supernatent so simply freeze your pellets until you know where your protein is. Protein purification from this supernatent, however, will depend on the properties of the protein: its isoelectric point (pI), size, hydrophobicity, etc.

http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3063

Protein Expression and Purification Protocol

From Plasmid to Protein

Step 1: Transform appropriate DNA plasmid into BL21(DE3) E. coli cells. These cells must be competent. (Protocol for how to make competent cells.)

a) Take competent cell stock aliquot (about 100 mL) out of -80oC freezer. Place on ice to thaw about 5 minutes.

b) Take one mL of plasmid DNA (usually a pRSET B vector with your protein cloned inside DNA seq of pRSET B) and add to the thawed competent cells. Vortex briefly to mix, and let incubate on ice for at least 20 minutes. Longer incubation will not hurt.

c) Heat shock the competent cells at exactly 42oC for 1 minute. Place on ice.

d) Add heat shocked cells to 1 mL of LB broth in a sterile culture tube, but DO NOT ADD antibiotic. Shake at 37oC for at least 1 hour. Shaking for 3 hours doesn't seem to hurt.

e) Plate the cell culture onto LB agar plates (usually 100 mL of culture will do although it depends on the competency of the cells and the initial concentration of plasmid). Make sure the plates have the appropriate antibiotic (usually ampicillin at 100 mg/ml concentration). (Protocol for how to make LB Ampicillin Agar Plates) Incubate the plate at 37oC for 14 hours (overnight generally). Longer times of incubations will allow for satellite colonies to appear.

Step 2: Make a starter culture for protein expression. Usually to about 250 to 500 mL of LB broth the antibiotic is added.

a) Generally, the antibiotic is ampicillin, which is added to the LB broth at 100 mg/ml final concentration. (Have a stock of ampicillin in the 4oC which is 1000x concentrated or 100 mg/ml in sterile distilled water to make things easier).

b) Pick a colony or two from your plate using a sterile innoculation loop and add to the LB broth/ampicillin flask. Incubate in a shaker (250 RPM) at 37oC until the OD @ 600 nm is 0.5 to 1.5 OD. (Anywhere in this range is fine, however, extremely dense starter cultures>1.5 OD can cause problems in protein expression. Under these dense growth conditions the plasmid seems to be rejected or turned-off by BL21(DE3)s.)

c) Save your starter culture flask by placing in the 4oC overnight. This will slow down the bacterial growth enough, but prevent the problems dense cultures cause as listed above.

Step 3: Make the big batch of bacteria culture for protein expression. (How many 1.5 L volumes should you grow up? Expect about 100 mgs of protein per liter, but that estimate is very dependent on the protein!)

a) Equally divide your starter culture among several 1.5 L volumes of sterile LB broth in 2.8 L Fernbach culture flasks. (Good precision when equally dividing the starter will better control the timing when induction can occur.)

b) Add antibiotic to 100 mg/ml final concentration. Grow them up in a New Brunswick shaker large enough to hold several Fernbach flasks.

c) Grow until an OD 600 nm of 0.8 to 1.2. Then induce the culture to express protein by adding 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. This is expensive stuff so use it carefully. Only measure out exactly the amount of IPTG you need for your flasks. Dissolve that amount in about 10 ml of sterile water and divide it equally among your 1.5 L flasks. This stage is called induction. Keep the culture shaking at 37oC.

d) Induction of protein generally takes 3 to 4 hours (but this too depends on your protein). After induction, centrifuge your bacteria in 500 ml bottles in the big Sorvall rotor at 5,000 RPM (bigger than the GSA found on the 2nd floor with Rice, Correl, and Moffat Labs). Do this in batches until all your culture is spun down saving the cell pastes each time. Freeze the cell paste at -80oC.

Step 4: Lysis and sonication of the bacteria. There are many ways to lyse bacteria, skin chickens etc., but this method is tried and true.

a) Make Lysis Buffer: 25 mM TRIS-Cl, 2 mM EDTA, pH 7.6. (You may add protease inhibitors like benzamidine or PMSF if you like but add these right before you start to lyse. Make concentrated PMSF solutions in pure ethanol since it is hydrophobic.) Then add lysozyme at 100 mg/ml concentration (or just a tipful from a spatula). Generally the volume lysis buffer is 1/20 to 1/50 the volume of the bacterial culture.

b) Resuspend the frozen cell paste as best you can in the Lysis Buffer using a 10 ml pipet or whatever means necessary. Let this suspension incubate for 20 minutes at room temperature, or until the suspension becomes turbid and viscous due to release of the bacteria's genomic DNA.

c) In order to eliminate the extreme turbidity of the suspension, sonicate the suspension to shear the DNA until the turbidity is similar to that of a normal protein solution. Then centrifuge at 18,000 RPM in a Sorvall SS-34 rotor (or ~40,000 x g) for 20 minutes at 4oC.

d) Save the pellet and the supernatent. (If the solution is slighly turbid due to residual DNA, a quick way to shear the DNA is to pass through a syringe with a needle.) Generally your protein is in the supernatent so simply freeze your pellets until you know where your protein is. Protein purification from this supernatent, however, will depend on the properties of the protein: its isoelectric point (pI), size, hydrophobicity, etc.

http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3063

pGEX derivatives from GE is used extensively for purification of GST-tagged proteins for immunization. U can also use the pET expression systems as described in the detailed protocol article below: http://www.ncbi.nlm.nih.gov/pubmed/18428660

Whether u need a large tag like GST or can use a small His tag depends on the solubility and nature of ur protein (GST makes proteins more soluble, but since the fusion tag is big, u can have GST-specific antibodies if u do not chop off the tag).

Good Luck

Thanks for your response. Best, Vlado

It's important to know whether this is a secreted or intracellular protein. S. aureus secretes lots of proteins, and purification from the medium may be the best way to isolate some of these. Also, many of these extracellular proteins have disulfide bonds, which won't form correctly during standard intracellular expression in E. coli.

If it is an intracellular protein, then recombinant expression in E. coli might be your best shot, as Manoj and Arvind mention. I'd advise you to look at Current Protocols (the big red binders of protocols that many labs have) for the details.

Be aware that S. aureus has a very AT rich genome, and E. coli is comparatively GC rich. That means that the codon usage of an S. aureus gene may not be well-optimized for expression in E. coli; see http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/optimisation_expression_levels/ for details.

There are strains of E. coli with extra copies of rare tRNAs which can help alleviate the codon-bias problem: Rosetta from Novagen and CodonPlus from Stratagene.

Thank you so much Wes. We are looking at intracellular protein and i think i will have to go with cloning. I will look at the suggestion you gave me. I really appreciate it.

Best regards,

Vlado

Thanks for  sharing knowledge.

Thanks for sharing knowledge.

Just seen the attached file. Many people have worked on this and even got theor Ph.D.s. You will find a host of such references on the net. However, I am attaching a file for you. It may not give you a direct or exact answer but will help to construct your answer for your question.