Ssm6a recombinant expression and purification method and used fusion protein
A fusion protein and purification tag technology, applied in the field of large-scale recombinant expression and purification of polypeptides, to achieve the effect of expanding production scale and saving production costs
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0125] Embodiment 1 genetic recombination engineering bacteria construction
[0126] Design the fusion expression sequence (SEQID NO:1) including thioredoxin-6×His-tag-enterokinase cleavage site-Ssm6a, and then optimize its coding gene sequence according to the codon preference of Escherichia coli to obtain the final optimization The final coding nucleotide sequence, SEQ ID NO: 2, was commissioned to be synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. (synthesis order number: 80-28327346).
[0127] The synthesized gene fragment was digested with Nde I and Xho I (Fermentas Company), and connected to the pET28a plasmid (product of Novagen Company, operated according to the manufacturer's suggestion) after double digestion with the same enzymes. The ligated recombinant pET28a plasmid was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing verification (sequence number: CX170710388-S), and the sequence was confirmed to be correct.
[0128] CaCl 2 Prepare Esc...
Embodiment 2
[0129] Embodiment 2 Recombinant Engineering Bacteria Shake Flask Fermentation
[0130] The strains finally obtained in Example 1 were inoculated into 100 ml LB medium (containing 50 μg / ml kanamycin) at 220 rpm (revolutions per minute), and cultivated overnight at 37° C. as seeds. Inoculate 10L of LB medium (containing kanamycin 50μg / ml) according to the volume ratio of 1% for fermentation (2L shake flask, liquid volume 1L, 10 bottles in total): first, 220rpm, 37°C to OD 600 After reaching 0.8, adjust the temperature to 30°C, add IPTG (final concentration 0.2mM) to induce protein expression for 8h (hours), and centrifuge at 8000rpm for 5min (minutes) to harvest the cells.
Embodiment 3
[0131] Embodiment 3 Recombinant Engineering Bacteria 15L Tank Fermentation
[0132] The strains finally obtained in Example 1 were inoculated into 320 ml LB medium (containing 50 μg / ml kanamycin), 220 rpm, and cultured overnight at 37° C. as seeds. The overnight seeds were inoculated in 8 L of LB medium (containing 50 μg / ml kanamycin) at a volume ratio of 4%. The initial temperature is 37°C, the initial rotation speed is 200rpm, the initial ventilation rate is 1:0.5, and the pH value is controlled to 6.8 by adding concentrated ammonia water throughout the process. Dissolved oxygen was maintained at 30%-40% by increasing the rotating speed, ventilation and adding glucose-supplemented medium (60% glucose, 2% magnesium sulfate). When OD 600 When it reaches 20°C, adjust the temperature to 30°C, add IPTG (final concentration 0.5mM) to induce protein expression, and finish the fermentation after 10 hours, and harvest the cells by centrifugation at 8000rpm for 5min.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More