This protocol will discuss the factors affecting plasmid expression efficiency in the Troubleshooting and Optimization of Inducible Promoter Expression Proteins sections. This experiment is from the next volume of the Molecular Cloning Laboratory Guide (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Expression of clonized genes in Escherichia coli using IPTG inducible promoter experiment
Materials and Instruments
IPTG-inducible expression vectors of E. coli strains suitable for transformation with lacIq or lacIq1 alleles Move makings For more product details, please visit Aladdin Scientific website.
IPTG SDS gel SDS-polyacrylamide gel Target gene or cDNA fragment LB agar plate LB medium
SorvallGSA turn-table or equivalent Boiling water bath Vibratory clamps
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)
1XSDS Gel Spiking Buffer
Store 1xSDS gel 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 preparation of SDS polyacrylamide gels for protein isolation.
Nucleic acids and oligonucleotides
Target gene or cDNA fragments
Medium
LB agar plates with ampicillin (50ug/ml)
LB medium with ampicillin (50ug/ml)
Centrifuges and rotors
SorvallGSA turntable or equivalent
Special equipment
Boiling water bath
Vibratory clamps
Additional 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
Transformation-ready E. coli strains with lacIq or lacIq1 alleles
IPTG-inducible expression plasmids with lacIq alleles (e.g. PMAL and pGEX) can transform any E. coli strain in the laboratory (e.g. JM101, DH5F', and TG1). See this program for details.
IPTG-inducible expression vectors
Other vectors include pGEM-3Z (Promega, see Figure 15-4), pGEX-1 (Pharmacia), pKK223-3 (Pharmacia), pMEX (U.S. Biochemicals), pTrc99A (Pharmacia) and pMAL ( NewEnglandBiolabs).
Positive control plasmids (IPTG-inducible vectors expressing LacZ fusion proteins of known size) 
Methods
Construction of E. coli strains containing recombinant expression vectors
1. PCR-modified or restriction endonuclease-digested isolated DNA fragments with restriction enzyme sites at the 5' and 3' ends that correspond to the IPTG-inducible expression vector.
Most IPTG-inducible expression vectors contain all the control elements necessary for expression of the exogenous protein. rCR modifies the expression cDNA/gene so that there are no extraneous sequences on either side of the structure. To facilitate expression, additional regulatory elements can be added at the end of the fragment depending on the vector used and preliminary results. Additional regulatory sequences are added at the end of the fragment (see Troubleshooting and Optimization of Inducible Promoter Expression Proteins in this protocol).
To be sure that the amplification reaction has not introduced false mutations, the PCR product should be analyzed for sequence.
2. DNA fragments containing the target CDNA/gene are ligated into the expression vector (Scheme 17 or 19 in Chapter 1).
3. The recombinant plasmid is transformed into an E. coli strain with the lacIq allele. If the plasmid itself has the lacI gene, any appropriate E. coli strain can be used. Spread the transformants on LB plates containing ampicillin (50ug/ml) and incubate at 37°C overnight.
Empty expression plasmid (negative control) and positive control plasmid transform interspersed E. coli strains.
4. Screening of transformants with inserted 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, leading to the formation of inclusion bodies that
5. 1~2 colonies of control and recombinant bacteria were picked out respectively, and connected to lml of LB culture medium containing ampicillin (50ug/ml), and incubated at appropriate temperature (20~37°C) overnight.
The growth rate of E. coli is 4 times slower at room temperature than at 37°C, so overnight incubation at ~20°C (~16 h) may not reach saturation. However, the slow metabolism of bacteria at low temperatures does not allow for the formation of inclusion bodies. See the Handling of Insoluble Proteins section in the introduction to this chapter.
6. Take 50ul of overnight culture into 5 ml of LB culture medium containing ampicillin (50ug/ml) and incubate at 20~37°C for more than 2 h with shaking until mid-logarithmic ( A550=0.5~1.0).
7. Pipette 1ml 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 mmol/L and continue to aerate at 20~37°C (see Optimization of IPTG Concentration and Induction Temperature below).
The concentration of IPTG has a strong influence on the expression level. 1mmol/L is just a starting point and a relatively isolated concentration. In the experiment, the IPTG concentration should be varied from 0.01 to 5.0 mmol/L to find the optimal concentration to use. For some proteins, it is necessary to induce slow transcription of the expression plasmid in order not to overload the bacterial biosynthesis system.
Probably the most important factor affecting the ability to obtain high levels of expression in E. coli is the growth temperature, and experimentally determining the optimal temperature. is critical for the expression of exogenous proteins. While successful expression has been obtained between 15 and 42°C, the optimal temperature range for expression of a particular protein may be as narrow as 2 to 4°C. The reasons why temperature sometimes plays a determining role in the level of expression, while at other times it has no effect on the level of expression, need to be further investigated, but may be the result of a number of factors acting either individually or in concert. These factors include: bacterial growth rate, intracellular folding of the expression product, availability of cofactors (heme, flavin, adenine dinucleotide, biotin, etc.), thermal denaturation of exogenous proteins, overloading of the cellular secretion or folding apparatus, the activity of endogenous proteases or other lytic enzymes, and activation of the bacterial SOS repair system. Because of these uncertainties, theoretical extrapolation of optimal growth temperatures is unreliable and repeated experiments must be performed.
9. At different times of induction (e.g., 1, 2, 4, and 6 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 of 1XSDS gel spiking buffer, heat at 100°C for 3 min, centrifuge at high speed for 1 min at room temperature and leave on ice until all samples are processed.
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 (please see Appendix 8).
A positive control induced at 37°C for 30 min has a glutathione transferase (GST) band with a molecular mass of 26kDa, and the amount of GST continues to rise during induction. Recombinant bacteria induced for a certain period of time should have a band of the expected size, and the kinetics of induction and protein stability may differ from the GST control.
Massive expression of target protein
14. Pick a recombinant E. coli colony into 50 ml of LB culture medium containing ampicillin (50ug/ml) and incubate in a 250 ml shake flask at 20-37°C overnight.
15. Take 5~50 ml of the overnight culture into 450~500 ml of LB culture medium containing ampicillin (50 ug/ml), and incubate in a 2L shake flask at 20~37°C with shaking, until mid-logarithmic ( A550=0.5~1.0).
16. The expression of target proteins was induced by 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 turntable) for 15 min at 4°C and the purification protocol continued later:
- If a GST fusion protein is expressed, proceed to protocol 5.
- If a GST fusion protein is expressed, proceed to protocol 5. If a maltose-binding protein fusion protein is expressed, proceed to protocol 6.
- If the expression product has a histidine tag, continue to Scheme 7 
