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 a bacteriophage T7 promoter expression plasmid (e.g., pET vectors).

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 E. coli strain BL21(DE3) or HMS174(DE3) with aliquots of the ligation reaction. Select for ampicillin-resistant transformants by plating aliquots of the transformation reaction on NZCYM agar plates containing 50 pg/ml ampicillin. Incubate the plates overnight at 37°C.

4. Screen the 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 (NZCYM medium containing 50 pg/ml ampicillin) with a transformed colony containing positive control vectors, negative control vectors, and one containing the recombinant vector. Incubate the cultures overnight at 37°C to obtain a saturated culture.

6. Inoculate 5 ml of NZCYM medium containing 50 pg/ml ampicillin in a 50-ml flask with 50 pl of a saturated culture. Incubate the cultures for 2 hours at 37°C.

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 culture (zero-time aliquot) to a microfuge tube. 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 1.0 mM and continue incubation at 20-37°C with aeration.

9. At 0.5, 1, 2, and 3 hours after induction, 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 pl of 1x SDS gel-loading buffer, and heat the samples to 100°C 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 pg 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 NZCYM containing 50 pg/ml ampicillin in a 250-ml flask with individual colonies of E. coli containing the recombinant and control plasmids. Incubate the cultures overnight at 37°C.

15. Inoculate 450-500 ml of NZCYM containing 50 pg/ml ampicillin in a 2-liter flask with 5-50 ml of overnight culture of E. coli containing the recombinant plasmid. Incubate the culture with shaking at 37°C 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 4°C 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

Was this article helpful?

0 0

Post a comment