Experiments on secretion and expression of exogenous proteins with alkaline phosphatase promoter and signaling sequences
Experiments on secretion and expression of exogenous proteins with alkaline phosphatase promoter and signaling sequences
This experiment describes the process of expressing exogenous proteins in Escherichia coli using the alkaline phosphatase promoter (phoA) and signaling sequences for secretion. This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Experiments on secretion and expression of exogenous proteins with alkaline phosphatase promoter and signaling sequences
Materials and Instruments
E. coli strain pTA1529 or pBAce Positive control plasmid Move makings For more product details, please visit Aladdin Scientific website.
Cauloblue staining solution or silver staining solution Micronutrient MOPS salts Neutral phosphate buffer SDS gel spiking buffer SDS-polyacrylamide gel Target gene or cDNA fragments Induction medium LB agar plates LB medium
Sorvall GSA turn-table or equivalent Sorvall SS-34 turn-table or equivalent Boiling water bath
Buffers and solutions
See 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.
Micronutrients (for 10xMOPS salts)
37 mg ( NH4)6Mo7O24-4H2O
84 mgCoCl2
158 mgMnCl2-4H20
247 mgH3BO3
25 mgCuSO4
18 mgZnSO4
Dissolve in 10 ml final volume, filter and sterilize, store at 4°C.
10XMOPS Salt (for induction medium)
400 mmol/L 3-(N-morpholino) propanesulfonic acid (pH 7T4) (MOPS)
40 mmol/L Wheat Flavonoids (pH7.4)
0.1 mmol/L FeSO4-7H2O
95 mmol/L NH4Cl
2.8 mmol/L K2SO4
5umol/LCaCl2-2H2O
5.3 mmol/LMgCl2-6H2O
0.5mol/NaCl
Dissolve in 1L final volume and filter for sterilization. Add 10ul of micronutrients per liter before use.
Neutral phosphate buffer (1 mol/L) (for induction medium)
1 mmol/L Na2HPO4 and 1 mmol/L NaH2PO4 equimolar infusion.
1XSDS gel spiking buffer
1xSDS gel spiking buffer without DTT was stored at room temperature, and 1 mol/LDTT storage solution was added to the above buffer as it was used.
Gel
SDS-polyacrylamide gel (10%)
Refer to Appendix 8 for SDS-polyacrylamide gel preparation for protein isolation.
Nucleic acids and oligonucleotides
Target gene or cDNA fragment
Medium
Induction Medium
1xMOPS salt (recipe above)
0.2% (m/V) glucose
0.2% (m/V) Casein hydrolysate (vitamin analytical grade; DIFCO Corporation)
20ug/ml Adenine
0.5ug/ml Thiamine
0.1mol/L neutral phosphate buffer (recipe above)
Mix 1 liter of aqueous solution from each storage solution, filter and sterilize and store at 4°C.
Low-phosphate medium for phoA induction (Neidhardt et al., 1974; originally used for isotope labeling), although troublesome to prepare, always results in the most aqueous induction.
LB agar plates containing ampicillin (50ug/ml) were used for the induction of phoA.
LB medium with ampicillin (50ug/ml)
Centrifuges and rotors
Sorvall GSA turntable or equivalent
Sorvall SS-34 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 Option 17 or 19 of Chapter 1.
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 strains
Any E. coli strain can be used as a host for vectors, and Oka et al. (1985) reported that the exogenous protein was expressed 5-fold higher in YK537 than in C600, which carries an internal deletion mutation such as the phoA gene, but the difference in the level of expression may not be due to the mutation because B.L. Wanner (personal communication) et al. found no difference in the expression of exogenous proteins in the homologous wild-type or phoA8 strains. There was no difference in the expression of the exogenous protein in the wild-type or phoA8 strains. If the exogenous protein was not toxic, a bacterial strain with a phoR mutation could be used. Because the wild-type phoR gene encodes a repressor of the manipulator, phoR mutant strains comprise expression/promoter-controlled genes (Wanner 1987). For all bacterial expression systems it may be necessary to try more than one type of host to find the E. coli strain that is best suited to express a particular exogenous protein (Weikert et al. 1988). Different strains should also be cultured at different temperatures to obtain the highest expression levels and protein stability (see Scheme 1).
The pTA1529 or pBAce
Plasmid pTAl529 was constructed by Oka et al. (1985); pBAce was constructed by C.C. Wang (Department of Pharmaceutical Chemistry, University of Caliafornia, SanFrancisco).
Positive control plasmid (IPTG inducible vector expressing LacZ fusion protein 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 corresponding to pTA1529 or pBAce at the 5' and 3' ends of the fragments.
To make sure that the amplification reaction does not introduce false mutations, the PCR product should be analyzed for sequence.
2. DNA fragments containing the target cDNA/gene are ligated into an expression vector (Scheme 17 or 19 in Chapter 1).
3. The ligated product is transformed into an appropriate E. coli strain. The transformants are spread on LB plates containing ampicillin (50ug/ml) and incubated at 37°C overnight.
The empty expression plasmid was transformed into the same E. coli strain as a 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. Pick 1~2 colonies of control and recombinant bacteria respectively, and insert them into lml of LB culture medium containing ampicillin (50ug/ml), and incubate at 37°C overnight.
6. Take 50ul into 5 ml of induction medium containing ampicillin (50ug/ml) and incubate at 20~37°C with shaking.
For factors affecting the induction of expression, please refer to the note in Step 8 of Protocol 1.
7. At different times after inoculation (e.g. 0, 6, 12, 18 and 24 h), take 1 ml of sample and put it into a microcentrifuge tube, determine the A550, and centrifuge it at high speed for 1 min at room temperature.
Induction of the phoA promoter is slower than the other promoters because induction occurs with the continuous depletion of phosphorus pitch in the medium. The optimal growth temperature also varies depending on the expressed protein and must be determined experimentally.
8. Sediment is suspended in 100ul lxSDS gel spiking buffer, heated at 100°C for 3 min, centrifuged at high speed at room temperature for 1 min, and placed on ice until all samples have been processed.
9. The sample is heated to room temperature and a suspension of 40ug or the equivalent of 0.15OD550 culture is applied to a 10% SDS polyacrylamide gel.
10.8~15V/cm electrophoresis until bromophenolan migrates to the bottom of the separator gel.
11. Observe bands of expression products by staining with komassie blue or silver, or by immunoblotting (see Appendix 8).
If a phoA signaling peptide is used to direct the secretion of exogenous proteins, carry out the Supplementary Scheme Subcellular localization of phoA fusion proteins, and analyze the distribution of proteins in the cell by hierarchical separation.
Bulk expression of target proteins
12. Pick a recombinant E. coli colony into 25 ml of LB culture medium containing ampicillin (50ug/ml) and incubate overnight in a 125 ml shake flask at 37°C with ventilation.
13.5000 g (6500r/min Sorvall SS-34 turntable) centrifugation for 15 min to collect the cells, and the precipitate was resuspended in 25 ml of induction medium and centrifuged again to collect the cells.
14. The washed cells were resuspended in 2.5 ml of induction medium, inoculated in 500 ml of induction medium, and incubated in 2L shake flasks at the optimal time and temperature determined in the pre-test.
15. After appropriate time of induction, the cells were collected by centrifugation at 4000 g (5000r/min Sorvall GSA head) at 4°C for 15 min.
If a phoA expression vector with a signal peptide sequence is used and the Supplementary Scheme results indicate that the exogenous protein is located in the periplasm. The expression product was purified by the osmotic shock method described in the Supplementary Scheme; if the vector used did not carry the signal peptide sequence, the exogenous protein was purified by conventional chromatographic methods. 
