Method for purifying recombinant lactase by using elastin like protein tag
An elastin-like and lactase-like technology is applied in the biological field to achieve the effect of high practical application value and simple method
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Embodiment 1
[0026] In this embodiment, the lactase gene tt412 is preferred. The recombinant lactase produced by this gene has high activity at low temperature, its optimum pH (7.0) is close to the pH value (6.7-6.8) of natural milk, and it is resistant to natural milk. Various ions (Na + 、K + , Ca 2+ etc.) and a series of advantages.
[0027] The prokaryotic expression vector is preferably the Escherichia coli expression vector pET-32a(+), which has a strong T7 promoter, so that the expression level of the foreign gene on the vector is very high, thereby facilitating the subsequent purification steps.
[0028] The preferred expression strain of Escherichia coli expression strain is BLR (DE3), which is recA - Derivatives of bacteriophage can increase the expression level of a single plasmid, and can stabilize plasmids with repetitive sequences or reduce the loss of DE3 prophage caused by the expression products of plasmids.
[0029] Step 1: Construct the lactase fusion expression vecto...
Embodiment 2
[0044] The processing steps of this embodiment are basically similar to those of Embodiment 1, and Step 1 is the same as that of Embodiment 1. The difference in other processing steps is also the difference in the specific parameters used:
[0045] (2) Transform the Escherichia coli expression strain BLR (DE3) by electroporation with the constructed expression vector, and then inoculate the identified transformant into a 250ml Erlenmeyer flask (containing 100ml TB medium), culture at 180rpm, 30°C until OD 600 reach 0.5, and then carry out self-induced expression at 150rpm and 25°C for 75h.
[0046] (3) The cells were collected by centrifugation at 6000g for 10 min at 6°C. The cells were washed once with 30ml, 50mM Tris buffer (pH8.0), and resuspended in 5ml, 50mM Tris buffer (pH8.0). Ultrasonic crushing (8W, 3s crushing time, interval 5s, total time 15min), centrifugation at 12000g for 18min to remove insoluble substances such as cell debris, and the crude enzyme solution was ...
Embodiment 3
[0053] The processing steps of this embodiment are basically similar to those of Embodiment 1, and Step 1 is the same as that of Embodiment 1. The difference in other processing steps is also the difference in the specific parameters used:
[0054] (2) Transform the Escherichia coli expression strain BLR(DE3) by electroporation with the constructed expression vector, and then inoculate the identified transformant into a 250ml Erlenmeyer flask (containing 100ml TB medium), culture at 250rpm, 37°C until OD 600 reach 0.6, and then carry out self-induced expression at 150rpm and 30°C for 65h.
[0055](3) Centrifuge at 10000g at 8°C for 10min to collect the bacteria, wash once with 30ml, 50mM Tris buffer (pH8.0), resuspend in 5ml, 50mM Tris buffer (pH8.0), and use Ultrasonic crushing (power 6W, crushing time of 4s, interval of 8s, total time of 20min), centrifugation at 14000g for 25min to remove insoluble substances such as cell debris, and the crude enzyme solution was placed in ...
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