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Protein product reuse method

A protein and product technology, which is applied in the field of protein product reuse, can solve the problems that the refolding process cannot restore the activity of recycling, reduce the degradation of protein chains, and the promotion effect is limited, so as to increase the number of reuse, simplify the process, increase the concentration and The effect on success rate

Inactive Publication Date: 2015-06-24
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to (1) molecular chaperones can only be used in the renaturation process, and the promotion effect on renaturation is limited and the cost is high; (2) in the normal use cycle of protein products, there is no protective effect, enzymatic hydrolysis and chemical After degradation, the renaturation process cannot restore activity and recycling. In contrast, PEG covalently modified protein products can effectively reduce the degradation of protein chains during the service life, and the denaturation-renaturation process does not require additives, and renaturation The concentration is high and the yield is significantly improved. Therefore, coupling PEG modification and refolding will be a new way to reuse protein products

Method used

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Effect test

Embodiment 1

[0038] Cultivation, refolding and purification of recombinant bacteria:

[0039] Strains were stored at -80°C. Scrape the surface of the frozen glycerol tube with a sterilized inoculation needle, then immediately scratch the bacteria adhering to the inoculation needle on the surface of the LB agar plate containing 100 μg / ml Amp, and incubate overnight at 37°C. Pick a single colony and inoculate them in three 500ml shake flasks containing 100ml LB culture medium respectively, culture at 37°C, shaker 240rpm, and transfer to 3L liquid LB medium when the measured OD600 reaches 1.0 (add 100μ / ml Amp ), cultivated under the same conditions, and when OD600 reached 1.0, it was transferred to a 100L fermenter as a secondary seed liquid. Ferment at 37°C, adjust rotation speed and ventilation during fermentation to keep oxygen saturation at 20%-60%, induce with 1mM IPTG when OD600 is 1.0 to 2.0, keep oxygen saturation at 20%-60%, pH In 7.0-7.4, (ammonia and hydrochloric acid adjustment)...

Embodiment 2

[0043] PEG modification uses Mal-mPEG modifier for fixed-point modification, the molecular weight of Mal-mPEG modifier is 20KD, the modification conditions are: the buffer uses Tris-HCl, pH8.5, the molar ratio of modifier to composite interferon is 10:1, and the modified The temperature is 4 degrees, and the modification time is 6 hours.

[0044] Equilibrate the chromatographic column (about 5 column volumes) with loading buffer (50mM Tris-HCl, pH8.5), take the supernatant after filtering the modified sample and put it on the anion exchange column, and the linear flow rate of loading is 4ml / min . After loading the sample, rinse with loading buffer (50mM Tris-HCl, pH8.5) at a linear flow rate of 4ml / min until the absorption at 280nm returns to the baseline. The modified sample was purified from 0-100% gradient with elution buffer (50mM Tris-HCl, 1M NaCl, pH8.5), the elution linear flow rate was 5ml / min, 10 column volumes were washed, and the elution peaks were collected respec...

Embodiment 3

[0046] According to the provisions of the Pharmacopoeia of the People's Republic of China (2005 Edition III), the activity and activity changes of the compound interferon and its PEG-modified products were detected at the cellular level by the cytopathic inhibition method. Principle: According to the effect that interferon can protect human amniotic cells (WISH) from vesicular stomatitis virus (VSV), the surviving human amniotic cells (WISH) were stained with crystal violet, and the absorbance was measured at a wavelength of 570nm. The protective effect curve of interferon on human amniotic cells (WISH) can be obtained, and the biological activity of interferon can be determined accordingly. When human amnion cells (WISH) are attacked by VSV virus, the reciprocal of the interferon dilution that can protect half of the cells from being attacked is 1 activity unit. Detect the initial value before denaturation-refolding operation, and the compound interferon activity is 5.44×10 ...

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Abstract

The invention relates to a protein product reuse method. The method includes the steps of: conducting PEG (polyethylene glycol) modification on protein, when the activity decreases and the product is out of the period of validity, subjecting the product to thorough denaturation treatment, and performing refolding. The method provided by the invention has the advantages of enhancing the times of reutilization and reducing the cost. Also, in the refolding step, the concentration and the success rate of renatured protein can be effectively improved. The method has the advantages of simple process, no special additive requirement, and low cost, can be used for reuse of protein products in daily chemical, industry and other aspects so as to deal with harsh working conditions and higher cost reduction demand.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for reusing protein products, which includes the steps of modifying the protein with PEG, after the activity is reduced, and after the product expires, the product is thoroughly denatured and refolded. The advantage is that the repeated use times of the protein product can be increased and the cost can be reduced. Moreover, in the refolding step, the concentration and success rate of refolding protein can be effectively increased. technical background [0002] Compared with chemical products, protein products are faced with the problem of unstable structure and rapid decrease in activity over time, which leads to short shelf life and high price of protein products. [0003] Some of this instability comes from physical factors, and some from chemical factors. The most common instability is that under certain conditions, the secondary, tertiary and quaternary stru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/113C07K1/18
Inventor 郑春杨王永宏冯建敏张琳王琳
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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