Extraction and purification method of nicotinamide ribose phosphate transferase
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A purification method and phosphoribose technology, applied in the field of bioengineering, can solve problems such as the difficulty of effectively separating and purifying genetic recombinant proteins, and achieve the effect of improving yield and purity
Pending Publication Date: 2021-11-23
XINTAI JIAHE BIOTECH CO LTD +1
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However, since the expression system determines the properties of the product during cell culture and the possible miscellaneous proteins, it has always been a difficult problem to effectively isolate and purify recombinant proteins from organisms
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Embodiment 1
[0034] Example 1: Production of fermented broth containing NAMPT enzyme
[0035] (1) Strain activation: Streak the low-temperature-preserved NAMPT enzyme production strain on an LB plate containing 100 μg / ml KAN and 100 μg / ml AMP, and culture at 33°C for 24 hours; 100μg / ml KAN and 100μg / ml AMP on LB plates, cultured at 33°C for 24h, set aside.
[0036] The medium formula of LB plate: peptone 10.0g, yeast powder 5.0g, NaCl 10.0g, agar 15.0g, water 1.0L.
[0037] (2) Preparation of primary seed liquid: use the inoculation loop to scrape the lawn of the production strain activated in the second step (1), inoculate it in LB liquid medium, 33°C, 200r / min, shake the flask for 12h to obtain the primary seed liquid.
[0038] LB liquid medium formula: peptone 10.0g, yeast powder 5.0g, NaCl 10.0g, water 1.0L.
[0039] (3) Preparation of secondary seed liquid: the primary seed liquid prepared in step (2) is inoculated into the seed tank for fermentation and cultivation according to th...
Embodiment 2
[0046] Embodiment 2: Extraction and purification of NAMPT enzyme
[0047] (1) The fermented liquid produced in Example 1 is centrifuged at 4000r / min for 15min, and the thallus in the fermented liquid is collected;
[0048] (2) Add pure water to the cells (999g of water per 1g of cells), adjust the pH to 7.2-7.4 with ammonia water, and centrifuge after passing through a homogenizer (homogeneous pressure 15,000PSI, homogeneous flow rate 400L / Hr) , centrifuged at 10000r / min, centrifuged for 20min, and separated to obtain the supernatant;
[0049] (3) The supernatant is decolorized by activated carbon and filtered through a ceramic membrane (the aperture of the ceramic membrane is 100nm, and the filtration pressure is 0.5MPa) to process the supernatant to obtain the filtrate;
[0050] (4) add nonionic detergent Triton X-100 and sodium chloride in filtrate, make the final mass concentration of Triton X-100 be 2%, the final concentration of sodium chloride is 500mM, remove inclusio...
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Abstract
The invention discloses an extraction and purification method of nicotinamide ribose phosphate transferase, which comprises the following steps: (1) carrying out fermentation culture on an NAMPT production strain to obtain fermentation liquor, centrifuging, and collecting thalli in the fermentation liquor; (2) adding pure water into the thalli, adjusting the pH value to 7.2-7.4 by using ammonia water, passing through a homogenizer, centrifuging, and separating to obtain supernate; (3) sequentially carrying out activated carbon decoloration and ceramic membrane filtration treatment on the supernate to obtain filtrate; (4) adding a nonionic detergent into the filtrate to remove inclusion bodies so as to obtain crude enzyme liquid; and (5) carrying out affinity chromatography on the crude enzyme liquid by adopting a nickel column, collecting eluent, carrying out electrodialysis desalination on the eluent, concentrating, crystallizing and drying, and extracting and purifying to obtain the NAMPT. By adopting the method disclosed by the invention, the NAMPT can be effectively extracted and purified from the fermentation liquor containing the nicotinamide phosphoribosyltransferase, and the yield and the purity of the NAMPT are remarkably improved.
Description
technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for extracting and purifying nicotinamide phosphoribosyltransferase. Background technique [0002] Nicotinamide adenine dinucleotide (NAD) plays a vital role in various cellular physiological processes, including gene expression, DNA repair, calcium ion mobilization, stress, apoptosis, metabolism, proliferation The bioenergetic state of NAD even determines the life and death of cells, so enzymes involved in NAD biosynthesis and metabolism have also become attractive targets in drug discovery for various diseases. Nicotinamide phosphoribosyl transferase (nicotinamide phosphoribosyl transferase, NAMPT) is the rate-limiting enzyme of the NAD salvage synthesis pathway, which participates in important processes such as cellular material and energy metabolism, protein modification, and DNA repair through the synthesis of NAD. [0003] Therefore, NAMPT has a wide range ...
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