- Plasmid construction for LZR-ELP , (EK)10-TAT-LZE and PAP
- Expression of co-expression plasmid coding LZR-ELP
- Purification of LZR-ELP
- Expression of plasmid coding (EK)10-TAT-LZE
- Purification of (EK)10-TAT-LZE
- Assemblying system
- Characterization of assemblying system
- Other details
Method
▶Plasmid construction for LZR-ELP , (EK)10-TAT-LZE and PAP:
In order to prevent his-tag on LZR-ELP from interfering with self-assembly of LZR-ELP, we provided DNA sequences coding LZR-ELP and PAP respectively to BGI to get co-expression plasmid so that we could purify LZR-ELP through high affinity interaction between LZR and LZE.
We also designed DNA sequence coding (EK)10-TAT-LZE and provided it to GENWIZ to get recombination plasmid.
▶Expression of co-expression plasmid coding LZR-ELP:
·Inoculate E.coli containing pET-28a(+) into LB solid medium containing 10mM kanamycin. Culture overnight at 30℃.
·Extraction and characterization of LZR-ELP plasmid:
1. Extract LZR-ELP plasmid in E.coli using TIANprep Mini Plasmid Kit.
2. Agarose gel electrophoresis.
·Plasmid transformation:
1. 3 μL plasmid and 200 μL E. coli were mixed in 1.5 mL EP tube.
2. Mixture were store on ice for 30 min.
3. Mixture were heat shocked at 42℃ for 90 s and then add 800 μL LB liquid culture medium to the EP tube on ice.
4. All tubes were cultured at 37℃ for 40 min.
5. Centrifuge(12000rpm, 1min) and abandon supernatant.
6. Use 200μL pipette to beat up the precipitate to equality.
7. Coat the bacterial liquid on LB solid culture medium and culture overnight at 37℃.
·After transformation, BL21 containing LZR-ELP plasmid was cultured overnight at 30℃.
·Select 2 colonies of LZR-ELP and extend them in 10 mL LB liquid medium with 10μL 100mM Kanamycin.
·Culture overnight at 30℃.
·Large aqueous protein expression was performed as follows:
1. 500 μL of 100 mM Kanamycin, 500 mL LB liquid culture medium and 10 mL bacterial liquid were mixed in 1000 mL conical flask.
2. Mixture was cultured for 2 hours at 37℃.
3. Measure the absorbance value at 600 nm using 200 μL bacterial liquid.
4. Store 1 mL bacterial liquid (before induction) in 4℃ refrigerator.
5. The rest were added in 200 μL of 1 M isopropyl β D-1-thiogalactopyranoside (IPTG) and then induced at 20℃ for 5 h.
6. Store 1 mL bacterial liquid (after induction) in 4℃ refrigerator.
7. The rest were moved into 500 mL centrifuge tubes and then centrifuged for 10 min at 8000 rpm.
8. Abandon the supernatant.
9. Beat the precipitation up by 200 mL 0.9% NaCl solution and mix it well, then centrifuge for 10 minutes at 8000 rpm and abandon the supernatant.
10. Beat the precipitation up by 10 mL 0.9% NaCl solution and mix it well, then wash the tubes by another 2 mL 0.9% NaCl solution.
11. Centrifuge (8000rpm, 10 min) and abandon the supernatant.
12. Store precipitation in -20℃ refrigerator.
Small aqueous protein expression was also performed in a similar way, except that the volume of each component was reduced to 2 % of the original and 4 mL bacterial liquid (after induction) were centrifuged for 10 min at 10000rpm. Collect precipitation to reserve.
1. Extract LZR-ELP plasmid in E.coli using TIANprep Mini Plasmid Kit.
2. Agarose gel electrophoresis.
1. 3 μL plasmid and 200 μL E. coli were mixed in 1.5 mL EP tube.
2. Mixture were store on ice for 30 min.
3. Mixture were heat shocked at 42℃ for 90 s and then add 800 μL LB liquid culture medium to the EP tube on ice.
4. All tubes were cultured at 37℃ for 40 min.
5. Centrifuge(12000rpm, 1min) and abandon supernatant.
6. Use 200μL pipette to beat up the precipitate to equality.
7. Coat the bacterial liquid on LB solid culture medium and culture overnight at 37℃.
1. 500 μL of 100 mM Kanamycin, 500 mL LB liquid culture medium and 10 mL bacterial liquid were mixed in 1000 mL conical flask.
2. Mixture was cultured for 2 hours at 37℃.
3. Measure the absorbance value at 600 nm using 200 μL bacterial liquid.
4. Store 1 mL bacterial liquid (before induction) in 4℃ refrigerator.
5. The rest were added in 200 μL of 1 M isopropyl β D-1-thiogalactopyranoside (IPTG) and then induced at 20℃ for 5 h.
6. Store 1 mL bacterial liquid (after induction) in 4℃ refrigerator.
7. The rest were moved into 500 mL centrifuge tubes and then centrifuged for 10 min at 8000 rpm.
8. Abandon the supernatant.
9. Beat the precipitation up by 200 mL 0.9% NaCl solution and mix it well, then centrifuge for 10 minutes at 8000 rpm and abandon the supernatant.
10. Beat the precipitation up by 10 mL 0.9% NaCl solution and mix it well, then wash the tubes by another 2 mL 0.9% NaCl solution.
11. Centrifuge (8000rpm, 10 min) and abandon the supernatant.
12. Store precipitation in -20℃ refrigerator.
Small aqueous protein expression was also performed in a similar way, except that the volume of each component was reduced to 2 % of the original and 4 mL bacterial liquid (after induction) were centrifuged for 10 min at 10000rpm. Collect precipitation to reserve.
·Bacteria disruption:
beat the precipitation up by 15 mL binding buffer (50mM Tris, 150mM NaCl, pH 7.9) for Ni2+-charged chromatography resin, disrupt on ice for 10 min, centrifuge (12000rpm, 20min) and collect supernatant, finishing the steps sequentially.
As for small aqueous protein expression, 3 mL binding buffer were added in each tube, disrupt the bacteria with cell cracker, centrifuge (10000 rpm, 20 min) and collect supernanant, finishing the steps sequentially.
·Pretreatment of Ni2+-charged chromatography resin:
1. Wash the resin with 100 mL of 500 mM iminazole
2. Wash the resin with MiliQ for 1 hour
3. Wash the resin with 50mL 100mM EDTA
4. Wash the resin with MiliQ for 2 hours
5. Wash the resin with 50mL 4%(w/v) NiSO4·6H2O
6. Wash the resin with MiliQ for 1 hour
7. Equilibrate the resin with 1 L Binding buffer (50mM Tris, 150mM NaCl, pH 7.9)
·Purification by Ni2+-charged chromatography resin
1. Immediately load the Supernatant onto the column. Adjust the flow rate to 10 column volumes per hour.
2. Wash the column with 6 column volumes of Binding buffer (pH 7.8).
3. Wash the column with 100mL 6M guanidine hydrochloride.
4. Elute the bound protein with 100mL 500mM Imidazole elution buffer. Collect 1mL fractions from the column, and monitor the A280 of each fraction.
5. Assay the fractions of interest for the presence of the polyhistidine-tagged protein by analyzing 30 µL aliquots by electrophoresis through a 10% SDS-polyacrylamide gel.
·Using deionized water dialyze the protein solution overnight.
·Centrifuge (10000 rpm, 20 min) and collect supernatant.
·Concentration were measured by 10μL aliquot as follows:
·Protein solution concentration was measured as follows:
1. Sample1: add 100 μL of 0.15 M NaCl to 1 mL Coomassie Brilliant Blue calibrated solution.
2. Sample2: add 10 μL protein sample and 90 μL of 0.15 M NaCl to 1 mL Coomassie Brilliant Blue calibrated solution. Detect the concentration of sample2 after 5 min accurately.
3. Detect the concentration of deionized water, sample1 and sample2, and write down the concentration named y1 and y2 respectively.
4. Use the formula, y2-y1=4.085x-0.029 (R2=0.999), to get the value of x.
5. Protein concentration(mg/mL)=10x/molecular of protein.
·The rest was freeze-dried to reserve.
beat the precipitation up by 15 mL binding buffer (50mM Tris, 150mM NaCl, pH 7.9) for Ni2+-charged chromatography resin, disrupt on ice for 10 min, centrifuge (12000rpm, 20min) and collect supernatant, finishing the steps sequentially.
As for small aqueous protein expression, 3 mL binding buffer were added in each tube, disrupt the bacteria with cell cracker, centrifuge (10000 rpm, 20 min) and collect supernanant, finishing the steps sequentially.
1. Wash the resin with 100 mL of 500 mM iminazole
2. Wash the resin with MiliQ for 1 hour
3. Wash the resin with 50mL 100mM EDTA
4. Wash the resin with MiliQ for 2 hours
5. Wash the resin with 50mL 4%(w/v) NiSO4·6H2O
6. Wash the resin with MiliQ for 1 hour
7. Equilibrate the resin with 1 L Binding buffer (50mM Tris, 150mM NaCl, pH 7.9)
1. Immediately load the Supernatant onto the column. Adjust the flow rate to 10 column volumes per hour.
2. Wash the column with 6 column volumes of Binding buffer (pH 7.8).
3. Wash the column with 100mL 6M guanidine hydrochloride.
4. Elute the bound protein with 100mL 500mM Imidazole elution buffer. Collect 1mL fractions from the column, and monitor the A280 of each fraction.
5. Assay the fractions of interest for the presence of the polyhistidine-tagged protein by analyzing 30 µL aliquots by electrophoresis through a 10% SDS-polyacrylamide gel.
1. Sample1: add 100 μL of 0.15 M NaCl to 1 mL Coomassie Brilliant Blue calibrated solution.
2. Sample2: add 10 μL protein sample and 90 μL of 0.15 M NaCl to 1 mL Coomassie Brilliant Blue calibrated solution. Detect the concentration of sample2 after 5 min accurately.
3. Detect the concentration of deionized water, sample1 and sample2, and write down the concentration named y1 and y2 respectively.
4. Use the formula, y2-y1=4.085x-0.029 (R2=0.999), to get the value of x.
5. Protein concentration(mg/mL)=10x/molecular of protein.
▶Expression of plasmid coding (EK)10-TAT-LZE:
·Inoculate E.coli containing pET-28a(+) into LB solid medium containing 10mM kanamycin. Culture overnight at 37℃.
·Extraction and characterization of (EK)10-TAT-LZE plasmid:
1. Extract (EK)10-TAT-LZE plasmid in E.coli using EasyPure® Plasmid MiniPrep Kit.
2. Agarose gel electrophoresis.
·Plasmid transformation was performed as described in Expression of co-expression plasmid codingLZR-ELP above.
·After transformation, BL21 containing (EK)10-TAT-LZE plasmid was cultured overnight at 37℃.
·Select 2 colonies of (EK)10-TAT-LZE and extend them in 10 mL LB liquid medium with 10μL 100mM Kanamycin.
·Culture overnight at 37℃.
·Large aqueous protein expression was performed as follows:
1. 500 μL of 100 mM Kanamycin, 500 mL LB liquid culture medium and 10 mL bacterial liquid were mixed in 1000 mL conical flask.
2. Mixture was cultured for 2 hours at 37℃.
3. Measure the absorbance value at 600 nm using 200 μL bacterial liquid.
4. Store 1 mL bacterial liquid (before induction) in 4℃ refrigerator.
5. The rest were added in 500 μL of 1 M (IPTG) and then induced at 37℃ for 5 h.
6. Store 1 mL bacterial liquid (after induction) in 4℃ refrigerator.
7. The rest was moved into 500 mL centrifuge tubes and then centrifuged for 10 min at 8000 rpm.
8. Abandon the supernatant.
9. Beat the precipitation up by 200 mL 0.9% NaCl solution and mix it well, then centrifuge for 10 minutes at 8000 rpm and abandon the supernatant.
10. Beat the precipitation up by 10 mL 0.9% NaCl solution and mix it well, then wash the tubes by another 2 mL 0.9% NaCl solution.
11. Centrifuge (8000rpm, 10 min) and abandon the supernatant.
12. Store precipitation in -20℃ refrigerator.
Small aqueous protein expression was also performed in a similar way, except that the volume of each component was reduced to 2 % of the original and 4 mL bacterial liquid (after induction) were centrifuged for 10 min at 10000rpm. Collect precipitation to reserve.
1. Extract (EK)10-TAT-LZE plasmid in E.coli using EasyPure® Plasmid MiniPrep Kit.
2. Agarose gel electrophoresis.
1. 500 μL of 100 mM Kanamycin, 500 mL LB liquid culture medium and 10 mL bacterial liquid were mixed in 1000 mL conical flask.
2. Mixture was cultured for 2 hours at 37℃.
3. Measure the absorbance value at 600 nm using 200 μL bacterial liquid.
4. Store 1 mL bacterial liquid (before induction) in 4℃ refrigerator.
5. The rest were added in 500 μL of 1 M (IPTG) and then induced at 37℃ for 5 h.
6. Store 1 mL bacterial liquid (after induction) in 4℃ refrigerator.
7. The rest was moved into 500 mL centrifuge tubes and then centrifuged for 10 min at 8000 rpm.
8. Abandon the supernatant.
9. Beat the precipitation up by 200 mL 0.9% NaCl solution and mix it well, then centrifuge for 10 minutes at 8000 rpm and abandon the supernatant.
10. Beat the precipitation up by 10 mL 0.9% NaCl solution and mix it well, then wash the tubes by another 2 mL 0.9% NaCl solution.
11. Centrifuge (8000rpm, 10 min) and abandon the supernatant.
12. Store precipitation in -20℃ refrigerator.
Small aqueous protein expression was also performed in a similar way, except that the volume of each component was reduced to 2 % of the original and 4 mL bacterial liquid (after induction) were centrifuged for 10 min at 10000rpm. Collect precipitation to reserve.
▶Purification of (EK)10-TAT-LZE
·Bacteria disruption was performed as described in Purification of LZR-ELP above.
·Pretreatment of Ni2+-charged chromatography resin was performed as described in Purification of LZR-ELP above.
·Purification by Ni2+-charged chromatography resin was performed:
1. Immediately load the Supernatant onto the column. Adjust the flow rate to 10 column volumes per hour.
2. Wash the column with 6 column volumes of Binding buffer (pH 7.8).
3. Wash the column with 100mL 6M guanidine hydrochloride.
4. Elute the bound protein with 100 mL Imidazole elution buffer at gradient concentration of 50mM, 100mM, 200mM and 500mM, respectively. Collect 1mL fractions from the column, and monitor the A280 of each fraction.
5. Assay the fractions of interest for the presence of the polyhistidine-tagged protein by analyzing 30 µL aliquots by electrophoresis through a 10% SDS-polyacrylamide gel.
·Protein samples were dialyzed and their concentration were measured as described in Purification of LZR-ELP above.
1. Immediately load the Supernatant onto the column. Adjust the flow rate to 10 column volumes per hour.
2. Wash the column with 6 column volumes of Binding buffer (pH 7.8).
3. Wash the column with 100mL 6M guanidine hydrochloride.
4. Elute the bound protein with 100 mL Imidazole elution buffer at gradient concentration of 50mM, 100mM, 200mM and 500mM, respectively. Collect 1mL fractions from the column, and monitor the A280 of each fraction.
5. Assay the fractions of interest for the presence of the polyhistidine-tagged protein by analyzing 30 µL aliquots by electrophoresis through a 10% SDS-polyacrylamide gel.
·See Labbook or Result for details
▶Characterization of assemblying system
·Sample preparation for TEM:
1. Pick up a piece of copper net with pointed tweezers, and the bright side of copper net face up.
2. Absorb 10 μL sample with a syringe. Press the copper net with the oblique side of the syringe and drop the sample into a ball on it. Be careful not to let the liquid touch the paper and do not let the copper net turn over.
3. Dry for 8-10 minutes.
4. Suck the liquid away with a pipette and dry for 1 min.
5. Add the same volume of dye (phospho-molybdic acid) in the same way as drop sample and absorb dye after 40 s, which is accumulated accurately.
6. Dry for the 5-6 min.
·Sample preparation for AFM:
1. The silicon chip was ultrasonicated with MiliQ for 20 min, then washed 3 times with MiliQ and finally dried with nitrogen.
2. 10μL sample was dropped on the silicon chip.
3. Suck back the sample in about 10 min and allow the silicon chip to dry naturally.
·LB liquid culture media (500 mL)
1. Add 5 g Tryptone, 5 g NaCl and 2.5 g Yeast Extract into 1000 mL conical flask.
2. Using 500mL deionized water dissolve the mixture.
3. Sterilize it by vacuum sterilizer.
·LB solid culture media (100 mL)
1. Add 1 g Tryptone, 1 g NaCl and 0.5 g Yeast Extract into 250 mL conical flask.
2. Dissolve the mixture by 100 mL deionized water, shake up and then add 1.5 g agar.
3. Sterilize the mixture by vacuum sterilizer(121℃, 20 min), and then cool to 50℃.
4. Add 100 μL 100 mM Kanamycin into the vacuum sterilizer, shake up and pour into culture dishes.
·Gel of agarose gel electrophoresis
1. Dissolve 0.2 g agarose in 25 mL 1*TAE.
2. Boil the mixture using microwave oven , then shake the conical flask to cool the mixture. Repeat until the agarose was totally dissolved.
3. Stand it at room temperature for about 5 min.
4. Add 0.2 μL EB into the conical flask. Shake up and added the mixture into the mold.
5. Wait the solidification of the gel for 15-20 min.
·Samples prepared for SDS-PAGE
1. Centrifuge for 2 minutes at 12000 rpm, abandon the supernatant.
2. Add 25 μL ddH2O, 6.5 μL loading buffer, metal bath at 95℃ for 20 min.
3. Centrifuge for 2 minutes at 12000 rpm.
4. 10 μL supernatant was used for electrophoresis.
·SDS-PAGE protein gel electrophoresis
1. Preparation of polyacrylamide gel.
2. Preparation of samples.
3. Electrophoresis conditions: 300 V, 18 mA for 2 h.
4. Coomassie brilliant blue staining for 1 h.
Decoloration for 16 h.
·Pretreatment of dialysis bag
1. Cut the dialysis bag into small segments of appropriate length (10-20cm).
2. The dialysis bag was boiled in 2% (m/V) NaHCO3 and 1mM EDTA (pH 8.0)for 10 min.
3. Thoroughly clean the dialysis bag with ddH2O.
4. The dialysis bag was boiled in 1mM EDTA (pH 8.0)for 10 min.
5. After cooling, store at 4℃ and ensure that it is always immersed in 40% ethanol.