Expression of cloned genes in Escherichia coli using the T7 phage promoter experiments
Expression of cloned genes in Escherichia coli using the T7 phage promoter experiments
Tabor and Richardson in 1985 and Studier and Moffatt in 1986 proposed a new expression system utilizing the T7 phage promoter, using transcriptional signals obtained from the T7 phage genome. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Expression of cloned genes in Escherichia coli using the T7 phage promoter experiments
Materials and Instruments
E. coli strain HMS174(DE3) or BL21(DE3) pET vector or equivalent Positive control plasmid Move makings For more product details, please visit Aladdin Scientific website.
Positive control plasmid Cauloblue staining solution or silver staining solution IPTG SDS gel spiking buffer SDS-polyacrylamide gel Target gene or cDNA fragments NZCYM agar plates NZCYM medium
SorvallGSA turn-table or equivalent Boiling water bath
Buffers and solutions
Refer to Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
Caulmers Brilliant Blue Staining Solution or Silver Staining Solution
See Appendix 8.
IPTG (1mol/L)
1X SDS gel spiking buffer
Store 1xSDS spiking buffer without DTT at room temperature and add 1mol/LDTT storage solution to the above buffer as it is used.
Gel
SDS-polyacrylamide gel (10%)
Refer to Appendix 8 for the preparation of SDS-polyacrylamide gels for protein isolation.
Nucleic acids and oligonucleotides
Target gene or cDNA fragment
Culture media
NZCYM agar plates with ampicillin (50ug/ml)
NZCYM medium with aminobenzyl (50ug/ml)
Centrifuges and rotors
SorvallGSA turntable or equivalent
Special equipment
Boiling water bath
Supplementary reagents
Step 1 of this program requires the reagents listed in Chapter 8, Program 7.
Step 2 of this protocol requires the reagents listed in Chapter 1, Option 17 or 19.
Step 3 of this protocol requires the reagents listed in Chapter 1, Options 23-26.
Step 4 of this protocol requires the reagents listed in Chapter 12, Option 3.
Vectors and Bacterial Strains
E. coli strain HMS174(DE3) or BL21(DE3)
pET vector or equivalent
For this series of vectors, please consult the Novagen catalog (or the web site www.novagen.com).
Positive control plasmids (e.g., T7 phage vectors expressing fusion proteins of known size)
Methods
Construction of E. coli strains containing recombinant expression vectors
1. PCR-modified or restriction endonuclease digestion of isolated DNA fragments with restriction enzyme sites at the 5' and 3' ends corresponding to T7 phage promoter expression plasmids (e.g., pET vectors; Studier et al. 1990).
To make sure that the amplification reaction does not introduce false mutations, the PCR product should be analyzed for sequence.
If necessary, a strong ribosome binding site can be placed upstream of the ATG of the cDNA/gene.
2. The DNA fragment containing the target cDNA/gene is ligated into an expression vector (Scheme 17 or 19 in Chapter 1).
3. The ligated product is transformed into E. coli strains HMS174 (DE3) or BL21 (DE3). The transformants are spread on NZCYM plates containing ampicillin (50ug/ml) and incubated at 37°C overnight.
Empty expression plasmids were transformed with the same E. coli strains as negative control.
4. Screen transformants with insert fragments by colony hybridization and/or restriction enzyme digestion analysis of small quantities of prepared plasmids, oligonucleotide hybridization, or sequence analysis (see Scheme 3 in Chapter 12).
Optimization of inducible target protein expression
Many studies have shown that the rate of cell growth severely affects the expression of exogenous proteins, so it is important to control the amount of inoculated bacteria, the duration of cell growth before induction, and the density of cells after induction. Excessive or excessive growth rates can overburden the bacterial synthesis system and lead to the formation of inclusion bodies.
5. 1~2 colonies of control and recombinant bacteria were picked separately, and connected to lml of NZCYM culture medium containing ampicillin (50ng/ml), and incubated at 37°C overnight until saturation.
6. 50ul of NZCYM culture medium containing ampicillin (50ug/ml) should be inserted into 5 ml of NZCYM culture medium and incubated at 37°C for 2 h in a 50 ml shake flask.
7. Pipette lml of uninduced culture into a microcentrifuge tube and process as described in steps 9 and 10 below.
8. Add IPTG to the remaining culture to a final concentration of 1 mnol/L and continue to incubate at 20-37°C under aeration.
In some systems, T7 phage RNA polymerase expression is regulated by a heat-induced promoter rather than an IPTG-induced promoter. See the note on Scheme 1, Step 8 for factors affecting induced expression.
9. At different times of induction (e.g., 0.5, 1, 2, and 3 h), take 1 ml of sample in a microcentrifuge tube, measure A550, and centrifuge at high speed for 1 min at room temperature.
10. Suspend the precipitate in 100ul lxSDS gel spiking buffer, heat at 100°C for 3 min, centrifuge at high speed at room temperature for 1 min, place on ice, and process all the samples.
11. Warm the sample to room temperature and apply 40ug or a suspension equivalent to 0.15OD550 culture to a 10% SDS polyacrylamide gel.
12.8~15V/cm electrophoresis until bromophenolan migrates to the bottom of the separator gel.
13. Observe bands of expression products by staining with komassie blue or silver, or by immunoblotting (see Appendix 8).
There should be no difference in protein band shape between untransformed E. coli cells and cells transformed with the empty vector. Recombinant bacteria that have been induced for a certain amount of time should have a band of the expected size.
Bulk expression of the target protein
14. Pick recombinant and control E. coli colonies into 50 ml of NZCYM culture medium containing ampicillin (50ug/ml) and incubate overnight at 37°C in 250 ml shake flasks.
15. 5~50 ml of the overnight culture was added to 450~500 ml of NZCYM culture medium containing ampicillin (50ug/ml) and incubated in 2L shake flasks at 37°C with shaking until mid-logarithmic ( A550=0.5~1.0).
16. The expression of target proteins was induced at the optimal IPTG concentration, optimal time and optimal temperature determined by the pre-test.
17. After the appropriate time of induction, cells were collected by centrifugation at 5000 g (5500r/min Sorvall GSA from the rotor head) for 15 min at 4°C and the purification protocol continued later:
- If a GST fusion protein is expressed, continue with protocol 5.
- If a GST fusion protein is expressed, continue with protocol 5 If a maltose-binding protein fusion protein is expressed, continue with protocol 6
- If the expression product has a histidine tag, continue to Scheme 7 
