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