Bacterial Hosts and Protein Studies Brian & Phuong
Expression Studies: Purpose To find cell strain that gives best expression of protein(s) you are studying at.
Bacterial Hosts Commonly Used for Expression Studies in Our Lab BL21-Gold (DE3) (plyss/plyse) BL21 (DE3) C+ RP BL21 (DE3) C+ RIL Rosetta (DE3)
Bacterial Host Genotypes Lack of Lon protease and OmpT protease Knock out of restriction enzyme dcm methylase modifies DNA so that DNA becomes resistant to endonuclease Gold Lack of endonuclease I (enda) Hte phenotype: Cells are better to accept ligated or large DNA plasmid
Codon Bias Different organisms show particular preferences for one of the several codons that encode the same amino acid. Fast-growing microorganisms like E. coli and yeast have no trnas for rare codons
Codon Frequency in E. coli
Codon Plus Strains BL21 (DE3) CodonPlus- RIL BL21 (DE3) CodonPlus- RP AGG/AGA (arginine), AUA (isoleucine) and CUA (leucine) AGG/AGA (arginine) and CCC (proline) Stratagene Stratagene Rosetta (DE3) AGG/AGA (arginine), CGG (arginine), AUA (isoleucine), CUA (leucine), CCC (proline), and GGA (glycine) Novagen
Expression Studies: Procedures Grow transformed cells in culture tube containing 8 ml of LB + appropriate antibiotic(s) over night Overnight in 37 o C shaker 8 ml LB + Antibiotic(s)
Expression Studies: Procedures cont. Measure the OD and calculate how much should be put into 10 ml LB + antibiotic(s) to get OD = 0.1 Take two 1-mL samples Cell culture 2X O/N OD =? OD = 0.1 10 ml LB + Antibiotic(s)
OD = 0.1 OD = 0.6
Expression Studies: Procedures cont. When OD=0.6, take another two 1-mL samples Add IPTG at C f of 1mM into cell cultures IPTG 4 hours 2X t=0 OD = 0.6
Expression Studies: Procedures cont. Take ~ 8 1-mL samples from cell culture 4 hours after IPTG induction Cell culture 4 hours after IPTG induction 8X t=4
Expression Studies: Procedures Final Samples taken at different time points would undergo whole cell lysate to extract the protein(s) out of cell The extracted protein(s) would be ran on the gel to see if specific protein gets expressed.
Cell Strain ONLY Found in PETC Strain Name: PET-C (staff) Genotype: MarK Sum + IrinA Ann1E Tung r
Solubility Study Main Idea: now that you have your protein expressed. in which solution is it (most) soluble in?
Solutions Common Ingredients: TRIS, NaCl, Glycerol L: LDAO N: Native U: Urea D: NP40 (Igepal) BB: Bug Buster 2U: 2x [Urea] Other: R & T
Urea & Bug Buster Urea: denatures protein; denaturization increases solubility of some proteins Bug Bugster: disrupts E. Coli membrane to allow the proteins to escape ; works whether or not protein is soluble or not
Let s Start! 6 pellets from Expression Study to each tube, add only one solution (100 microliters) Mix (vortex) -> SONICATE Pellet + 100 microliters of 1 sol.
Max Speed ~ 5 mins
DON T FORGET TO LABEL EVERYTHING! X 6
Pellet--- Done with set aside Supernatent: need to separate the protein if it dissolved in the solution add acetone (1 milliliter) -Vortex -Ice -Spin - Remove acetone -37* incubator
Acetone GONE! Protein Present..Where? Add 25 x OD (taken from Expression Study) of 1x SLB-BME to both centrifuges and pellets (microliters) Heat Run Gel RUN SPOT RUN!
Solubility Study (Cont.) Where s the protein? Was it in the pellet or supernaten? Locate protein. If it was not soluble (was in the pellet). on to the next step
IFold Purpose Carefully extract the target protein and refold the protein if it was denatured by the solution (ie urea)
IFold Series of detergent and buffer washes to purify the protein. Inclusion bodies can be easily washed out by TCEP or LS [Tris(2-carboxyethyl)phosphine] N-Lauroylsarcosine
Protein is purified. Now let s find the best condition for refolding. IFold includes 92 different refolding conditions and 4 controls Each environment is unique, containing a mixture of different: ph levels, salt, cyclodextrin, redox agent, and refolding additives.
Add a sample of your protein to each of the wells. IFold comes with own matrix with the solutions ready to be used. Solutions must be kept frozen and thawed when needed.
After protein has had time to refold itself (done at room temperature), perform assays to determine which environment worked best Can measure refolding process by measuring absorbance
Perform assays: Got Protein? How well did your protein show up? Did it refold properly so that it works? Hopefully you ended with
Merci. Irina- for explaining to Phuong how expression study works.. Like a dozen times Mark- for putting up with our pestering questions that early morning Annie- for always hinting us toward the right answers but never giving it straight forward Sum- for all the computer stuff. Phuong doesn t work well with computers. Tung- Oh shoot I forget.. What was IFold again? Lisa, Joyce, Winnie- various part of the presentation and answering all of our random questions