Exercise 4 (continued) GENE EXPRESSION Introduction: Expression systems are designed to produce many copies of a desired protein within a host cell. In order to accomplish this, an expression vector is inserted into a host cell. This vector contains all of the genetic coding necessary to produce the protein, including a promoter appropriate to the host cell, a sequence which terminates transcription, and a sequence which codes for ribosome binding. One expression system was developed in 1986 by W. F. Studier and B. A. Moffatt, who created an RNA polymerase expression system which was highly selective for bacteriophage T7 RNA polymerase. This expression system is commonly known as the T7 expression system. The pET series of vectors have been developed for cloning and expression of recombinant proteins using the T7 system. These plasmids contain a T7 promoter which is specific to only T7 RNA polymerase (not bacterial RNA polymerase), a polylinker to clone in DNA, an antibiotic resistance gene, and a ColE1 origin of replication. The pET-44 vectors are designed for cloning and expression of peptide sequences fused with the Nus protein which has great solubility potential (recall the importance of correct folding). This vector encodes a Nus protein with a N-terminal His-tag to facilitate purification by metal chelate chromatography. Attached is a map for the pET-44 vector series. Without insertion of foreign DNA, the recombinant Nus protein expressed from pET-44 is ~68 kD. As indicated above, the T7 expression system depends on the regulated expression of T7 RNA polymerase, an extremely active enzyme that is encoded in the DNA of bacteriophage T7. The T7 RNA polymerase transcribes DNA beginning within a specific 23-bp promoter sequence called the T7 promoter. Copies of the T7 promoter are located at several sites on the T7 genome, but none is present in E. coli chromosomal DNA. In this expression system, recombinant E. coli cells have been engineered to carry the gene encoding T7 RNA polymerase next to the lac promoter. Typically, the host cell used is E. coli strain BL21(DE3). These cells then are transformed with plasmid vectors that carry a copy of the T7 promoter and, adjacent to it, the DNA encoding the desired protein (see Figure below). When lactose or a molecule similar to lactose, such as IPTG (isopropyl-ẞ-D-thiogalactopyranoside), is added to the culture medium containing these transformed, recombinant E. coli cells, T7 RNA polymerase is expressed by transcription from the lac promoter. The polymerase then binds to the T7 promoter on the plasmid expression vectors, catalyzing transcription of the inserted DNA at a high rate. Since each E. coli cell contains many copies of the expression vector, large amounts of mRNA corresponding to the cloned DNA can be produced in this system. Typically, 10 – 50 percent of the total protein synthesized by these cells after addition of IPTG is the protein of interest. Very high levels of Nus protein lac promoter Multiple T7 RNA polymerases T7 RNA Nus DNA polymerase gene T7 late promoter PET44 Recombinant E. coli chromosome Plasmid expression vector Figure – T7 Expression System - E. coli BL21(DE3) containing pET-44. 41 DAY 1 (REVIEW) Transformation into Expression Host BL21(DE3) For transforming into the expression host BL21(DE3), use 1-2 µl of the pET-44 plasmid (50 ng/μl) and follow your transformation procedure. After the heat shock and dilution, spread cells on LB and LB-Amp plates and incubate overnight at 37°C. Using 100 ng of pET-44 with the competent BL21(DE3) cells, approximately 500 colonies should be obtained per plate with your transformation procedure. Setting up for Option 3 below: Hint: Put this in your notebook. It is Ok to do this at home. You will be provided an ampicillin stock of 100 mg per ml. Write out the calculations for 5 ml LB containing 100 µg per ml of ampicillin, 50 ml of LB containing 100 µg per ml of ampicillin in an Erlenmeyer flask in your notebook. Make these solutions and store them in the refrigerator. DAY 2 Growth and induction: The following protocol is a “suggested/general" protocol. We will modify the protocol as needed; based on how the labs proceed. Write out the protocol that you actually use in your lab notebook. Make sure you save samples at each stage of the purification protocol. You will run all these samples on a gel. IT IS IMPERATIVE THAT YOU SAVE SAMPLES AT EACH STEP TO RUN ON AN SDS GEL. OPTIONS: We will use Option 3: 1) Pick a single colony from the plate of transformed cells and inoculate into 50 ml LB containing 100 μg per ml of ampicillin in a 250 ml Erlenmeyer flask. 2) Alternatively, streak a LB-Amp plate from a glycerol stock of BL21(DE3) cells containing pET-44, incubate overnight at 37°C, and inoculate a single colony into LB media containing ampicillin as above. 3) We will use this method: Pick a single colony from the plate of transformed cells and inoculate into 5 ml LB containing 100 µg per ml of ampicillin, incubate overnight at 37°C. Use this culture to inoculate 50 ml of LB containing 100 µg per ml of ampicillin in an Erlenmeyer flask. a) Pick a single colony from the plate of transformed cells and inoculate into 5 ml LB containing 100 µg per ml of ampicillin, incubate overnight at 37°C. (This will be performed by the lab technician). Transfer the 5ml of culture to the 50 ml flask. Incubate with shaking (100-150 rpm, depends on flask size) at 37°C until OD 600 reaches 0.5-0.7 (about 3-4 hours). (Sample size for O.D reading: 4 ml). Remove ~1.0 ml sample for the uninduced control, centrifuge, and store cell pellet at 0 to - 20°C. You may take the 1 ml from the 4 ml sample you removed for the OD reading. An alternate procedure (which we will not use in this class) place flask in 37°C oven (stationary) overnight and shake the following morning until the appropriate OD 600 is obtained. b) Chill the cells on ice for 15-30 minutes. c) Add IPTG to a final concentration of 0.2mM (100µl of 100mM IPTG (provided) per 50ml of culture). 42 d) Continue shaking at overnight at room temperature at 100 rpm. An alternate procedure (which we will not use in this class) is to shake the cells at 100 rpm@ 37°C for 3-4 hr. Efficient IPTG induction results in reduction of cell growth. Post-induced cultures are typically less than 2X the initial cell density. (Record OD600) e) Following induction, remove ~1.0 ml sample, centrifuge, and store pellet at 0 to -20°C for analysis of total cell protein. You may take the 1 ml from the 4 ml sample you removed for the OD reading. f) Place the flask of the post-induced culture on ice for 10 min or store the cells in the refrigerator. Harvest the cells by centrifugation at 5000xg for 5-10 min at 4°C. Resuspend the cells in 1-1.5 ml of 50mM Tris-Cl (pH 8.0), transfer to eppendorf tube, and centrifuge for ~1 min. Remove the supernatant and store the cells at -20°C or continue with lysis and purification. Protein extraction is typically more effective with frozen cells. DAY 3 Lysis If cells have been stored at -20°C, remove from freezer and thaw at room temperature for 10-15 min. a) For a cell pellet harvested from 40-50 ml of culture, resuspend in 1 ml of Bacterial Lysis Reagent by gently pipetting up and down until the cell suspension is homogenous. Rotate the tube for an additional 10 min at room temperature. Alternatively, invert tube to mix cell suspension every couple min over 10 min. Do not vortex. b) Centrifuge at 12,000 rpm for 8 min to separate soluble and insoluble fractions. The soluble protein is in the supernatant. c) Transfer the supernatant to a clean tube (Soluble Lysate #1). Save for purification. d) Repeat extraction by resuspending the insoluble fraction in 1 ml of Bacterial Lysis Reagent. Gently pipet up and down until suspension is homogeneous. Centrifuge at 12,000 rpm for 8 min e) Transfer the supernatant to a clean tube (Soluble Lysate #2). Save for purification. f) Save 40 μl aliquots of Soluble Lysates #1 and #2 for SDS-PAGE analysis. You will only use 20 ul for the gel on Day 4. This gives you some extra sample in case you need to rerun the gel for any reason. Metal Chelate Affinity Purification Buffers (included) for Metal Chelate Chromatography Binding Buffer: 20mM Tris-Cl (pH 8), 0.5M NaCl, 5mM imidazole Wash Buffer: 20mM Tris-Cl (pH 8), 0.5M NaCl, 20mM imidazole Elution Buffer: 20mM Tris-Cl (pH 8), 0.5M NaCl, 200mM imidazole The metal chelate chromatography columns contain a resin with nickel-nitrilotriacetic acid (Ni-NTA) coupled to cellulose beads. The Ni-NTA matrix binds proteins carrying a stretch of at least six consecutive histidine residues. The Nus protein expressed from pET44 carries such a His-tag sequence to allow affinity purification via the nickel ion bound to the resin matrix. The target protein is recovered by elution with imidazole. 43 The metal chelation chromatography columns are ready to use for rapid purification of His-tagged proteins. Each column is packed with ~0.4 ml of metal chelate resin containing 20% ethanol as preservative. In general, the binding capacity is ~5 mg protein per ml of resin. - 1) In an eppendorf storage rack, place six tubes (12 x 75 mm, included) and three eppendorf tubes (2 ml, included). Label sequentially the 12 x 75 mm tubes – Equilibration, Unbound Soluble Lysate #1, Unbound Soluble Lysate #2, Wash 1, Wash 2, and Wash 3. Label the eppendorf tubes as Elute 1, Elute 2, and Elute 3. 2) Snap off the bottom tip of a column and place in Equilibration tube. Remove top cap and allow the excess packing buffer to drain by gravity to top of gel bed. If column does not begin to flow, push cap back into top of column and remove. If column still does not begin to flow, attach syringe (included) to bottom of column and gently pull out the syringe to start flow. Place column back into tube to drain. 3) Discard drained buffer and place column back into Equilibration tube. Hint: Centrifuge both Soluble Lysate 1 and Soluble Lysate 2 at 12,000 rpm for 8 min (AGAIN). This is to avoid adding any cell debris to the column which will slow the column down. 4) Apply 0.75 ml of Binding Buffer and allow column to drain. Repeat with 0.75 ml of Binding Buffer. 5) Place column into next tube and carefully apply sample from Soluble Lysate #1. Allow sample to fully drain into collection tube. Hint: it is better to leave a little liquid behind than add debris to the column. 6) Remove column, place into next tube, and apply sample from Soluble Lysate #2. Hint: it is better to leave a little liquid behind than add debris to the column 7) After sample has fully drained, remove column and wash three times with 1 ml of Wash Buffer, collecting the flow-through fractions in Wash 1 -3 collection tubes. 8) Following the last wash, place column into first eppendorf tube and elute with 250 μl of Elution Buffer (Remember this would contain your purified product- SAVE IT! Remove 40 ul for gel analysis). 9) Repeat with 400 µl of Elution Buffer, collecting eluate in tube labeled Elute 2 (Remember this would contain your purified product- SAVE IT! Remove 40 ul for gel analysis). 10) Repeat with 400 μl of Elution Buffer, collecting eluate in tube labeled Elute 3 (Remember this would contain your purified product- SAVE IT! Remove 40 ul for gel analysis). DAY 4 SDS-PAGE Analysis Analyze column fractions on 7.5-10% SDS gels. The Nus protein expressed from pET-44 runs at ~68 kD. Some degradation of the purified Nus protein will be observed due to absence of protease inhibitors. Protein assays will not be run for this lab, however if protein assays are performed on elution fractions, load 5-10 ug. Most of the protein eluted from the Ni-NTA column will be in the Elute 2 fraction (1-2 µg/µl). If protein assays are not performed, run ~10µl from Elute 1, 2 and 3. Loading the gel: i. If total cell protein from uninduced and induced cells is also to be analyzed, resuspend cell pellets (1 ml cell cultures) in 80 µl Laemmli buffer and load 20 µl. 44 ii. For the other column fractions being analyzed, Soluble Lysate 1 and Soluble Lysate (15 μl) add 15 µl of Laemmli buffer. Load 30 μl of each sample on the gel. iii. For Elute 1, 2 and 3, take 15 µl samples and add 15 µl Laemmli buffer. Load 30 µl of each sample on the gel. iv. Do not forget to load a Kaleidoscope marker/ladder. It is OK to load marker in two lanes if available. It is sometimes useful to heat all the samples to 95°C for 5 minutes prior to loading. Coomassie Colloidal Blue Staining of SDS gels The Colloidal Blue Staining Reagent uses the colloidal properties of Coomassie G-250 dye for protein staining of polyacrylamide gels. The reagent stains only protein and allows bands to be viewed directly during the staining process. Standard Procedure 1) Following SDS-PAGE, remove gel and place in clean dish. Rinse gel three times with 100 ml of deionized water (5 min incubation each wash). 2) Mix the Colloidal Blue Stain Reagent just before use by gently inverting and swirling the reagent bottle. 3) Discard water wash and add 20-30 ml of the Colloidal Blue Stain Reagent to the gel. Gently shake or periodically agitate. Stain intensity should reach maximum within 2 hr. Gels may be stained overnight without increasing background. 4) If desired, destain with 100 ml of water for 1-2 hrs. Alternative Procedure for Faster Staining: We will use this procedure 1) Following SDS-PAGE, place gel in clean dish. Rinse gel twice with 100 ml water pre-heated to 70-90°C (5 min each wash). 2) Mix the Colloidal Blue Stain Reagent just before use by gently inverting and swirling the reagent bottle. The instructor will do this step and pre-heat the dye for you. 3) Discard water wash from gel and add 20-30 ml of the pre-heated Colloidal Blue Stain Reagent. Protein bands should be visible within 5-10 min. Destain gel with 100 ml of water for 10-20 min. You may take a picture immediately or leave your gel in water in the refrigerator till the next class. Hint: This is data, it should go in your notebook. Alternate procedures (which we will not use in class): Microwave gel plus stain for 1 min or until solution begins to boil or put the tray containing the gel and stain reagent may be placed on a hot plate and heated until the solution begins to boil. Remove from hot plate. Protein bands should be visible within 5-10 min. If desired, destain gel with 100 ml of water for 10-20 min. References: This document has been adapted from “Bacterial Transformation” http://plaid.hawk.prattsburgh.edu/faculty/slsh/tranformation.html Transformation and gene expression protocol has been adapted from the procedure suggested by Protein Express, Inc. 45 45