This experiment was mainly used to obtain soluble active proteins.
Operation method
Purification of expressed proteins from inclusion bodies
Principle
High levels of protein expression in E. coli often result in the formation of cytoplasmic granules or inclusion bodies visible under phase contrast microscopy. These inclusion bodies, which are aggregated from expressed proteins, are easily separated from soluble and membrane-bound proteins. Bacteria expressing high levels of exogenous proteins are concentrated by centrifugation and can be lysed mechanically, by sonication, or by lysozyme plus detergent. After precipitation by centrifugation, inclusion bodies can be washed with Triton X-100 and EDTA or with urea. In most cases, adjusting the washing conditions will result in a purity of more than 90% of the exogenous protein in the inclusion bodies.
Materials and Instruments
E. coli cells expressing target proteins Move makings Caveat Inclusion body recharacterization precautions1. The optimal pH range is between 8.0 and 9.0;2. The temperature should be 4℃;3. The protein concentration should not be too large, generally 0.1-0.2mg/mL. 4;4. the recovery time is generally 24-36 hours;5. low molecular compounds, such as L-Arg, can help increase the solubility of complex intermediates; urea, guanidine hydrochloride, alkylurea, and carbonic acid amides, etc., are very effective promoters in non-denaturing concentration, which can prevent protein aggregation; Tris has a promotional effect on protein complexation; EDTA can prevent protein degradation;6. the first thing is to obtain a higher purity of inclusion bodies;7. Inclusion body dissolution should be thorough, generally should make the dissolved liquid volume is large, do not be afraid of protein dilution, to help dissolution measures;8. centrifugation should be done both before and after dialysis9. Inclusion bodies should be washed thoroughly, and appropriate amount of Triton-X100 should be added to the washing solution;10. Dissolve the inclusion bodies and place them in a dialysis bag to be reconstituted in the reconstitution solution. 11;11. After reconstitution, dialyze slowly for 12-24h in a low concentration of buffer solution;12. The complexation rate is determined according to the optical properties of denatured and non-denatured proteins. 12. TritionX100 and SDS are descaling agents that are not easy to remove in the end and are generally not allowed in the pharmaceutical industry. Some inclusion bodies do not wash well with Triton. Inclusion body washing is a very important step in purification, change the culture conditions, and then wash the inclusion bodies, sometimes only 3-4 stray bands can be left before the column, so the subsequent purification is very convenient.14. The concentration of protein at the time of reconstitution is generally 0.1-1mg/ml, too high concentration is easy to form polymer precipitation, too low concentration is not economical, and many proteins are unstable at low concentration and easily denatured. Common Problems I. Additional programs, please refer to http://img.dxycdn.com/trademd/upload/userfiles/image/2015/09/B1442910480png_small.jpg II. Protein refolding methods, please http://img.dxycdn.com/trademd/upload/userfiles/image/2015/09/A1442910608png_small.jpg III. Analysis of common problems of inclusion body recharacterization 1. Principles of inclusion body recharacterization Low concentration, gentle gradient, low temperature. 4 n( O5 ~0 q8 D;) 2. How to wash the inclusion bodies? The usual washing method is generally not clean, you can first dissolve the inclusion body with 6M guanidine hydrochloride, filter to remove the undissolved material, pay attention to the sample run electrophoresis, and then diluted to 4M, centrifugation of the precipitate and supernatant run electrophoresis, and so on can be diluted to the appropriate concentration, you can find a suitable way to remove the impurities, in fact, this is the method of gradient sedimentation, which is more effective than the usual direct elution. This is actually the gradient precipitation method, which is more effective than the usual direct elution. 3. How to choose between urea and guanidine hydrochloride? Urea and guanidine hydrochloride are medium-strength denaturants that are easily removed by dialysis and ultrafiltration. They have strong reversible denaturing effect on the hydrogen bond of inclusion body, the required concentration of urea 8-10M, guanidine hydrochloride 6-8M. urea dissolves inclusion body slower and weaker than guanidine hydrochloride, the solubility is 70-90%, urea will be cleaved to form cyanate when the action time is longer or the temperature is higher, and it will modifies the amino group of recombinant protein covalently, but the dissolution of urea is non-ionizing and neutral, the cost is low, and it will not cause a large amount of protein to be removed after denaturation. However, solubilization with urea has the advantages of non-ionization, neutrality, low cost, protein recovery without causing a large amount of protein precipitation, as well as solubilized inclusion bodies can be used for purification by a variety of chromatographic methods, and so on, so it has been widely used. Guanidine hydrochloride has a solubilization capacity of more than 95%, and it solubilizes quickly without causing covalent modification of recombinant proteins. However, it also has the disadvantages of high cost, easy to produce precipitation under acidic conditions, may cause a large amount of protein precipitation after removal after restitution, and interference with protein ion exchange chromatography. 4. How should the 8M urea solubilized inclusion body solution be stored? Half a month at 4 degrees Celsius is not a problem. Leaving the solution at room temperature for more than 48 hours may affect the target protein because urea can acylate some amino acids under alkaline conditions, so it is better to treat the BI solution earlier. 5. What is the protein concentration for reconstitution? Generally the concentration is 0.1-1mg/ml, too high concentration is easy to form polymer precipitation, too low concentration is not economical, and many proteins are unstable at low concentration, it is easy to denature. 6. Low concentration of protein after denaturation Proteins may have been degraded in the process of complexation. You can concentrate the compounded protein and run the gel to see. The complexation process is generally a low concentration of protein, need to ensure that there is enough folding space between the molecules. Some of the protein is not folded correctly in the precipitation, may not be able to see the precipitation, after the complexation of the protein high-speed centrifugation to see. 7. What is the problem of protein precipitation during replication? What should I do? If the protein precipitates out, it must be because the conditions have changed too drastically. For example, according to the composition of the solution of inclusion bodies, a solution should be configured every 1 PH or concentration value, and gradually dialyzed to normal. In addition, the dialysis must be performed at very low concentrations and under mild conditions to allow the protein to fold correctly. However the rate of denaturation should be very low. If the denaturant urea can be added to 2M, guanidine hydrochloride can be added to 1-1.5M; in addition, the concentration of glycerol can be increased to ≤30%, and the appropriate amount of glycerol can be added to the denaturing sample. 8. Detection of complexation effect According to the specific protein properties and needs, the complexation efficiency of protein can be detected from biochemical, immunological and physical properties. (1) Gel electrophoresis: Generally, non-denaturing polyacrylamide gel electrophoresis can be used which can detect proteins in denatured and natural states, or non-reducing polyacrylamide electrophoresis can be used to detect the pairing of disulfide bonds after the complexation of proteins with disulfide bonds. (2) Spectroscopic methods: ultraviolet difference spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy (CD), etc., can be used to detect the complexation situation by utilizing the spectroscopic features of the two states, but they are generally only used for process detection in complexation studies. (3) Chromatographic methods: such as IEX, RP-HPLC, CE, etc., due to the different chromatographic behaviors of the proteins in the two states. (4) Biological activity and specific activity assay: generally measured by cellular methods or biochemical methods, which better reflect the activity of complex proteins, it is worth noting that different methods of measuring activity measure different results, and often do not fully reflect the activity in vivo. (5) Viscosity and turbidity measurement: the solubility of the complexed protein increases, and in the denatured state, due to the exposure of hydrophobic residues, the water solubility is generally very poor, and most of them form a visible precipitate precipitation. (6) Immunological methods: such as ELISA, WESTERN, etc., especially the antibody test for structural determinant clusters, which reflects the folding state of the protein more realistically. For more product details, please visit Aladdin Scientific website.
Cell Lysis Buffer I Cell Lysis Buffer II Deoxycholic Acid Concentrated Hydrochloric Acid Inclusion Body Lysis Buffer I Inclusion Body Lysis Buffer II KOH PMSF SDS Gel Spiking Buffer Tris-Cl DNaseI Lysozyme with Urea SDS-Polyacrylamide Gel
Sorvall GSA turntable or equivalent pH paper Polished glass rod
Buffers and solutions
See Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
Cell Lysis Buffer I
50 mmol/LTris-Cl (pH 8.0)
1 mmol/LEDTA (pH 8.0)
100 mmol/LNaCl
Cell lysis buffer II, ice-cold
50 mmol/L Tris-Cl (pH 8.0)
10 mmol/L EDTA (pH 8.0)
100 mmol/L NaCl
0.5% TritonX-100
Deoxycholic acid
Use protein grade cholic acid/degreaser.
HCl (12 mol/L) (concentrated hydrochloric acid)
Inclusion body lysis buffer I
50 mmol/L Tris-Cl (pH 8.0)
1 mmol/LEDTA (pH 8.0)
100 mmol/LNaCl
8 mol/L urea
0.1mol/LPMSF
The buffer was prepared ready to use.
Inclusion Body Lysis Buffer II
50 mmol/LKH2PO4 (pH10.7)
1 mmol/LEDTA(pH8.0)
50 mmol/LNaCl
KOH(10mol/L)
PMSF (100 mmol/L)
1x and 2X SDS gel spiking buffer
DTT-free 1x and 2xSDS gel spiking buffers were stored at room temperature, and lmol/LDTT storage solution was added to the above buffers as it was used.
Urea-containing Tris-Cl (0.1mol/L, pH 8.5)
For use in Method 2 only, see Step 7. Prepare 0.1mol/L Tris-Cl (pH 8.5) containing increasing concentrations of urea (e.g., 0.5, 1, 2, and 5 mol/L). Use solid urea ready to use before use; do not use any urea storage solution, as urea tends to decompose.
Enzymes and buffers
DNaseI (1 mg/ml)
Lysozyme (10 mg/ml)
Prepared with Tris-Cl (PH 8.0) ready to use.
Gel
SDS-polyacrylamide gel (10%)
Refer to Appendix 8 for preparation of SDS-polyacrylamide gels for protein isolation.
Centrifuge and rotor head
Sorvall GSA turntable or equivalent
Special equipment
pH test paper
See step 10 for additional supplies.
Polished glass rod
Carrier and Bacterial Strain
E. coli cells expressing target proteins
Cultivate 1L of E. coli cells expressing the target protein as inclusion bodies
Methods
Preparation of cell extract
1.1L of expressed cell culture was centrifuged at 5000 g (5500r/min Sorvall GSA head) for 15 min at 4°C in a pre-weighed centrifuge tube.
Note: Steps 2 to 4 should be performed at 4°C.
2. Aspirate off the supernatant, weigh the sediment of the organisms and add 3 ml of Cell Lysis Buffer I per gram (wet weight) of organisms and gently swirl or stir with a polished glass rod to suspend the organisms.
3. Add 4ul of 100 mmol/L PMSF and 80ul of 10 mg/ml lysozyme per gram (wet weight) of organisms and stir for 20 min. Other protease inhibitor mixtures may also be added (see Protocol 1).
4. Add 4 mg deoxycholic acid per gram (wet weight) of organisms and continue stirring.
5. Leave the suspension at 37°C, stirring occasionally with a polished glass rod, and add 20ul of 1 mg/ml DNaseI per gram (wet weight) of organisms when it becomes viscous.
6. Leave the lysate at room temperature until it is no longer viscous (about 30 min).
Purification and washing of inclusion bodies
7. Purify and wash the inclusion bodies using one of the two methods described below.
Method 1: Extract the inclusion bodies with Triton X-100.
The following procedure is a modification of the method used by Marston et al (1984).
a. Cell lysates are centrifuged at 4°C for 15 min.
b. Remove supernatant by decanting and resuspend the precipitate in a 9-fold volume of 4°C Cell Lysis Buffer II.
c. Allow the suspension to stand at room temperature for 5 min.
d. Centrifuge at high speed for 15 min.
e. Decant the supernatant and set aside. Resuspend precipitate in 100ul of water.
f. Take 10ul of each supernatant and precipitate, mix with 10ul of 2XSDS gel spiking buffer, and analyze the distribution of target protein by SDS-polyacrylamide gel electrophoresis.
g. Continue with Step 8 to dissolve inclusion bodies if necessary.
Method 2: Extraction of inclusion bodies with urea
The following procedure for washing and lysing inclusion bodies with buffers containing different concentrations of urea is taken from Schoner et al. (1985).
a. Cell lysates were centrifuged at 4°C for 15 min at high speed.
Note: Steps b, d and f must be performed at 4°C.
b. Remove supernatant by decanting and resuspend each gram (wet weight) of bacterial precipitate in 1 ml of water. Fill 4 centrifuge tubes with 100ul each and store the rest at 4°C.
c. Centrifuge at high speed for 15 min at 4°C.
d. Each precipitate is resuspended in 100ul of 0.1mol/L Tris-Cl (pH 8.5) containing various concentrations of urea (e.g. 0.5, 1, 2 and 5mol/L).
e. High-speed centrifugation at 4°C for 15 min.
f. Decant the supernatant, set aside, and resuspend each bacillus precipitate in 100ul of water.
g. Take 10ul of each supernatant and precipitate and mix them with 10ul of 2XSDS gel spiking buffer respectively. SDS-polyacrylamide gel electrophoresis was performed to analyze which concentration of urea washes best.
h. Using the optimal concentration determined in step g, wash the remaining inclusion body precipitate according to the above protocol (step b).
i. Continue with Step 8 to dissolve the inclusion bodies if necessary.
Dissolving Inclusion Bodies
8. Take an appropriate amount of the resuspended cell precipitate from Step 7, centrifuge at high speed at 4°C for 15 min, and resuspend the precipitate in 100ul of Inclusion Body Lysis Buffer I containing 0.1 mmol/LPMSF (add as needed).
9. Leave at room temperature for lh.
10. Add the solution to 9 times the volume of Inclusion Body Lysis Buffer II and leave at room temperature for 30 min. check that the pH is maintained at 10.7 and adjust with 10mol/LKOH if necessary.
11. Adjust pH to 8.0 with 12mol/L hydrochloric acid and allow to stand at room temperature for at least 30 minutes.
12. Centrifuge the solution at high speed for 15 min at room temperature.
13. Decant the supernatant, set aside, and resuspend the precipitate in 100ul of 1XSDS gel spiking buffer.
14. Mix 10ul of supernatant with 10ul of 2xSDS gel spiking buffer, perform SDS polyacrylamide gel electrophoresis of the supernatant and precipitate, analyze the degree of dissolution, and continue with additional protocols. 
