Method for purifying Cas12a protein
A protein and buffer technology, applied in the biological field, can solve the problems of reduced protein effective yield, complex operation, protein decomposition, etc., and achieve the effects of reducing protein loss, improving protein purity, and reducing purification costs.
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Embodiment 1
[0030] Example 1: Construction of Cas12a protein expression vector and expression of Cas12a protein
[0031] Competent cells Rosseta (DE3) are taken and melted on ice. Take 1 μL of plasmid 6His-MBP-TEV-huLbCpf1 (Addgene) which has been inserted into the Cas12a coding sequence and add it to 50 μL competent cells, ice bath for 30 min, then heat excite at 42 °C for 45 s, quickly and smoothly put back into ice for 2 min, add 700 μL of sterile medium without antibiotics, mix well, shake at 37 °C for 1 h, pipette about 50 μL of bacterial liquid and spread evenly to contain 100 mg / L ampicillin and 30 mg / L ampicillin. L-chloramphenicol in LB agar medium plates, culture at 37 °C overnight. Colonies were picked and transferred to media containing 100 mg / L ampicillin and 30 mg / L chloramphenicol for overnight incubation.
[0032] Take 25 mL of saturated bacterial solution and add to 1 L containing 100 mg / L ampicillin and 30 mg / L chloramphenicol and 0.2% glucose medium, culture at 37 °C to an ...
Embodiment 2
[0033] Example 2: Extraction of Cas12a protein
[0034] Thaw the bacterial bodies on ice, resuspend the bacterial bodies according to the 1:4-5 volume of lysis buffer (50 mM Tris-HCl, pH = 8.0, 20 mM imidazole, 300 mM NaCl, 0.5 mM TCEP, 0.25 mg / ml lysozyme, 1:100 protease inhibitor), and then sonically broken under ice water bath conditions, ultrasound amplitude 40, total working time of 12 min. After sonication, centrifuge 40,000 g at high speed for 1.5 h, aspirate the supernatant, and retain a small amount of samples for subsequent protein electrophoresis. The supernatant is used for the purification experiment below.
Embodiment 3
[0035] Example 3: Purification of Cas12a protein using two nickel columns and heparin columns
[0036] The following buffers were used during purification:
[0037]Buffer A (50 mM Tris-HCl, pH = 8.0, 20 mM imidazole, 300 mM NaCl, 0.5 mM TCEP);
[0038] Buffer B (50 mM Tris-HCl, pH = 8.0, 300 mM imidazole, 300 mM NaCl, 0.5 mM TCEP);
[0039] Buffer C (20 mM HEPES, 0.5 mM TCEP, 100 mM NaCl);
[0040] Buffer D (2 M NaCl, 20 mM HEPES, 0.5 mM TCEP, pH =8);
[0041] Buffer E (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10% (v / v) glycerol).
[0042] The specific purification steps are as follows:
[0043] (1) The nickel column is crude and pure
[0044] First use peristaltic pump water to wash 5 column volumes, and then use buffer A to balance Hisrap HP 5 column volumes, and then use peristaltic pump to load the supernatant obtained in Example 2, the loading amount is about 100 ~ 150mL, and retain the flow through for electrophoresis, after the completion of the sample, first wash 5 to 10 colu...
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