Method for realizing purification of recombinant protein by using antifreeze protein-intein as purification tag and application
An antifreeze protein and purification tag technology, applied in the field of separation and purification of exogenous recombinant proteins, can solve the problems of increased cost of protease, separation steps, complicated process, difficulty in scale, etc., and achieve easy molecular cloning operation, simple operation and labor saving. Effect
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[0047] Example 1 Construction of pET-24a-GST-Mxe GyrA-linker-AFP expression vector
[0048] (1) Synthesize the following primers respectively:
[0049] SEQ ID NO.4: Primer P1:
[0050] 5’-GGAATTC CATATG ATGTCCCCTATACTAGGTTATTG-3', where the underlined part is QuickCut TM Nde I recognition site.
[0051] SEQ ID NO. 5: Primer P2:
[0052] 5’TGCATCTCCCGTGATGCA CATTCGCAT TTTTGGAGGATGGTCGCCACCACCAAAC-3', wherein the underlined part is the base sequence corresponding to the three amino acids MRM.
[0053] SEQ ID NO. 6: Primer P3:
[0054] 5’-GTTTGGTGGTGGCGACCATCCTCCAAAA ATGCGAATG TGCATCACGGGGAGATGCA-3', wherein the underlined part is the base sequence corresponding to the three amino acids MRM.
[0055] SEQ ID NO. 7: Primer P4:
[0056] 5’-ATCACTC AAGCTT AGCGTGGCTGACGAACCCGTTC-3', where the underlined part is QuickCut TM Hind III recognition site.
[0057] (2) Under the guidance of the above P1 and P2 primers, the GST gene was amplified by PCR using the pGEX-4T-1 pla...
Example Embodiment
[0059] Example 2 Heterologous expression of GST-Mxe GyrA-linker-AFP
[0060] The recombinant expression plasmid pET24a-GST-Mxe GyrA-linker-AFP obtained in Example 1 was transferred into E. coli BL21 (DE3) competent cells by heat shock method, and a single colony containing 50 μg / mL kanamycin was picked. Cultured in 5mL LB medium of 37°C for 12h, then transferred to TB medium containing 50μg / mL kanamycin at a ratio of 2% (v / v), and continued to culture at 37°C to OD 600 When it is 0.6 to 0.8, IPTG is added to a final concentration of 0.5 mM. After induction at 16 °C for 20 h, the bacterial cells were collected and resuspended in 50 mM Tris-HCl buffer pH 8.5 for sonication, followed by centrifugation, and the supernatant was collected for SDS-PAGE electrophoresis.
Example Embodiment
[0061] Example 3 Separation and purification of GST using antifreeze protein-intein tag
[0062] Preparation of ice shell: Measure 100mL of pre-cooled deionized water and pour it into a 500mL round-bottomed flask, immerse the round-bottom flask in the freezing solution and rotate quickly for 1min to make part of the deionized water in the round-bottom flask form a close contact with the round-bottomed flask. For the ice shell on the inner wall, pour out the remaining deionized water, continue to immerse the round-bottomed flask with the ice shell in the ice adsorption freezing solution and rotate for 2 minutes, until the ice shell appears evenly distributed cracks, and then the follow-up experiments can be carried out.
[0063] The cell disruption supernatant was diluted to 1 mg / mL, pre-cooled with ice-water mixture, and then slowly added dropwise to the surface of the ice shell in the round-bottomed flask, such as figure 2 As shown, connect the round-bottomed flask to the ro...
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