IPTG诱导的原核表达(英文).doc

IPTG诱导的原核表达Chapter 15, Protocol 1Expression of Cloned Genes in E. coli Using IPTG-inducible Promoters This protocol describes how (1) to clone cloned This protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying IPTG-inducible promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. .CAUTION.RECIPE.MATERIALSBuffers and SolutionsCoomassie Brilliant Blue stain or Silver stain. IPTG (1 M). 1x SDS gel-loading buffer. .Nucleic Acids and OligonucleotidesGene or cDNA fragment of interest .MediaLB agar plates containing 50 g/ml ampicillin. LB medium containing 50 g/ml ampicillin. .Additional ReagentsStep 1 of this protocol requires the reagents listed in Chapter 8, Protocol 7. Step 2 of this protocol requires the reagents listed in Chapter 1, Protocol 17 or Chapter 1, Protocol 19. Step 3 of this protocol requires the reagents listed in Chapter 1, Protocol 23 to Chapter 1, Protocol 26. Step 4 of this protocol requires the reagents listed in Chapter 12, Protocol 3. .Vectors and Bacterial StrainsE. coli strain suitable for transformation and carrying either the lacIq or lacIq1 allele Some IPTG-inducible expression vectors carry the lacIq allele on the expression plasmid (e.g., pMAL and pGEX). These can be used in any laboratory strain of E. coli (e.g., JM101, DH5F, and TG1).IPTG-inducible expression vector Other examples include pGEM-3Z (Promega), pGEX-1 (Pharmacia), pKK223-3 (Pharmacia), pMEX (U.S. Biochemicals), pTrc 99A (Pharmacia), and pMAL (New England Biolabs).Positive control plasmid (e.g., an IPTG-inducible vector known to express a LacZ fusion protein of defined size) .METHOD 1.Modify by PCR (Chapter 8, Protocol 7), or isolate by restriction enzyme digestion, a fragment of DNA carrying 5- and 3-restriction enzyme sites compatible with sites in an IPTG-inducible expression vector.Constructs generated by PCR should be sequenced to ensure that no spurious mutations were introduced during the amplification reactions. 2.Ligate the DNA fragment containing the cDNA/gene of interest into the expression vector (Chapter 1, Protocol 17 or Chapter 1, Protocol 19). 3.Transform an E. coli strain containing the lacIq allele with the recombinant plasmid. If the plasmid vector itself carries the lacI gene, then any appropriate strain of E. coli can be used. Plate aliquots of the transformation reaction on LB agar containing 50 g/ml ampicillin. Incubate the cultures overnight at 37C. 4.Screen transformants by colony hybridization and/or restriction enzyme analysis, oligonucleotide hybridization, or direct DNA sequence analysis (please see Chapter 12, Protocol 3) of plasmid minipreparations. 5.Inoculate 1-ml cultures (LB medium containing 50 g/ml ampicillin) with 1 or 2 colonies containing the empty expression vector, the positive control plasmid (pGEX-1), and the recombinant expression plasmid. Incubate the cultures overnight at the appropriate temperature (20-37C). 6.Inoculate 5 ml of LB medium containing 50 g/ml ampicillin with 50 l of each overnight culture. Incubate the cultures for 2 hours at 20-37C in a shaking incubator until cells reach mid-log growth (A550 of 0.5-1.0).It is important to monitor the number of bacteria inoculated into the growth medium, the length of time cells are grown before induction, and the density to which cells are grown after induction. 7.Transfer 1 ml of each uninduced culture (zero-time aliquot) to microfuge tubes. Immediately process the zero-time aliquots as described in Steps 9 and 10. 8.Induce the remainder of each culture by adding IPTG to a final concentration of 1 mM and continue incubation at 20-37C with aeration.But please see the notes below Step 8 in the print version of the manual concerning optimization of the IPTG concentration and the induction temperature). 9.At various time points during the induction period (e.g., 1, 2, 4, and 6 hours), transfer 1 ml of each culture to a microfuge tube, measure the A550 in a spectrophotometer, and centrifuge the tubes at maximum speed for 1 minute at room temperature in a microfuge. Remove the supernatants by aspiration. 10.Resuspend each pellet in 100 l of 1x SDS gel-loading buffer, and heat the samples to 100C for 3 minutes. Centrifuge the tubes at maximum speed for 1 minute at room temperature in a microfuge, and store them on ice until all of the samples are collected and ready to load on a gel. 11.Warm the samples to room temperature and load 0.15 OD550 units (of original culture) or 40 g of each suspension on a 10% SDS-polyacrylamide gel. 12.Run the gel at 8-15 V/cm until the bromophenol blue reaches the bottom of the resolving gel. 13.Stain the gel with Coomassie Brilliant Blue or silver, or carry out an immunoblot to visualize the induced protein. 14.For large-scale expression and purification of the target protein, inoculate 50 ml of LB containing 50 g/ml ampicillin in a 250-ml flask with a colony of E. coli containing the recombinant construct. Incubate the culture overnight at 20-37C. 15.Inoculate 450-500 ml of LB containing 50 g/ml ampicillin in a 2-liter flask with 5-50 ml of overnight culture of E. coli. Incubate with shaking at 20-37C until the culture has reached the mid-log phase of growth (A550 = 0.5-1.0). 16.Induce expression of the target protein based on the optimal values of IPTG concentration, incubation time, and incubation temperature determined in the previous section. 17.After the induced cells have grown for the proper length of time, harvest the cells by centrifugation at 5000g (5500 rpm in a Sorvall GSA rotor) for 15 minutes at 4C and proceed with a purification protocol: .Chapter 15, Protocol 5 if the expressed protein is a fusion with glutathione S-transferase.Chapter 15, Protocol 6 if the expressed protein is a fusion with maltose-binding protein.Chapter 15, Protocol 7 if the expressed protein contains a polyhistidine tag.RECIPES. .Coomassie.Dissolve 0.25 g of Coomassie Brilliant Blue R-250 in 90 ml of methanol:H2O (1:1, v/v) and 10 ml of glacial acetic acid. Filter the solution through a Whatman No. 1 filter to remove any particulate matter. Store at room temperature.Glycerol.To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biology-grade glycerol in 9 volumes of sterile pure H2O. Sterilize the solution by passing it through a prerinsed 0.22-m filter. Store in 200-ml aliquots at 4C.IPTG.IPTG is isopropylthio- -D-galactoside. Make a 20% (w/v, 0.8 M) solution of IPTG by dissolving 2 g of IPTG in 8 ml of distilled H2O. Adjust the volume of the solution to 10 ml with H2O and sterilize by passing it through a 0.22-m disposable filter. Dispense the solution into 1-ml aliquots and store them at -20C.LB.deionized H2O, to 950 ml.tryptone, 10 g.yeast extract, 5 g.NaCl, 10 g.For solid medium, please see Media Containing Agar or Agarose.To prepare LB (Luria-Bertani medium), shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (approx. 0.2 ml). Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle.Media Containing Agar or Agarose.Prepare liquid media according to the recipes given. Just before autoclaving, add one of the following:Bacto Agar (for plates).15 g/literBacto Agar (for top agar).7 g/literagarose (for plates).15 g/literagarose (for top agarose).7 g/literSterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. When the medium is removed from the autoclave, swirl it gently to distribute the melted agar or agarose evenly throughout the solution. Be careful! The fluid may be superheated and may boil over when swirled. Allow the medium to cool to 50-60C before adding thermolabile substances (e.g., antibiotics). To avoid producing air bubbles, mix the medium by swirling. Plates can then be poured directly from the flask; allow approx. 30-35 ml of medium per 90-mm plate. To remove bubbles from medium in the plate, flame the surface of the medium with a Bunsen burner before the agar or agarose hardens. Set up a color code (e.g., two red stripes for LB-ampicillin plates; one black stripe for LB plates, etc.) and mark the edges of the plates with the appropriate colored markers.When the medium has hardened completely, invert the plates and store them at 4C until needed. The plates should be removed from storage 1-2 hours before they are used. If the plates are fresh, they will sweat when incubated at 37C. When this condensation drops on the agar/agarose surface, it allows bacterial colonies or bacteriophage plaques to spread and increases the chances of cross-contamination. This problem can be avoided by wiping off the condensation from the lids of the plates and then incubating the plates for several hours at 37C in an inverted position before they are used. Alternatively, remove the liquid by shaking the lid with a single, quick motion. To minimize the possibility of contamination, hold the open plate in an inverted position while removing the liquid from the lid.NaCl.To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H2O. Adjust the volume to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl solution at room temperature.SDS.Also called sodium lauryl sulfate. To prepare a 20% (w/v) solution, dissolve 200 g of electrophoresis-grade SDS in 900 ml of H2O. Heat to 68C and stir with a magnetic stirrer to assist dissolution. If necessary, adjust the pH to 7.2 by adding a few drops of concentrated HCl. Adjust the volume to 1 liter with H2O. Store at room temperature. Sterilization is not necessary. Do not autoclave.SDS Gel-loading Buffer.100 mM Tris-Cl (pH 6.8).4% (w/v) SDS (electrophoresis grade).0.2% (w/v) bromophenol blue.20% (v/v) glycerol.200 mM dithiothreitol or. -mercaptoethanol.For a 2x buffer.1x and 2x SDS gel-loading b