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EP-4737472-A1 - METHOD FOR REMOVING PLANT-DERIVED SOLUBLE PROTEIN FROM PLANT-DERIVED EXTRACT

EP4737472A1EP 4737472 A1EP4737472 A1EP 4737472A1EP-4737472-A1

Abstract

The present invention provides a method for removing a plant-derived soluble protein from a plant-derived extract, the method comprising the following steps: (1) a step for insolubilizing the plant-derived soluble protein by freezing and thawing the plant-derived extract, and (2) a step for removing the insolubilized protein from the extract.

Inventors

  • MATSUO, KOUKI
  • MATSUSHIMA, AKITO
  • NISHIDA, NAOKO

Assignees

  • National Institute Of Advanced Industrial Science and Technology
  • Chiyoda Corporation

Dates

Publication Date
20260506
Application Date
20240628

Claims (10)

  1. A method for removing a plant-derived soluble protein from a plant-derived extract, the method comprising the following steps: (1) a step for insolubilizing the plant-derived soluble protein by freezing and thawing the plant-derived extract, and (2) a step for removing the insolubilized protein from the extract.
  2. The method of Claim 1, wherein the plant-derived soluble protein comprises RuBisCO.
  3. The method of Claim 1 or 2, wherein the plant is a plant belonging to the genus Nicotiana.
  4. The method of Claim 3, wherein the plant belonging to the genus Nicotiana is Nicotiana benthamiana .
  5. The method of Claim 1 or 2, wherein the plant expresses an exogenous soluble protein which is not insolubilized by freezing and thawing.
  6. The method of Claim 1 or 2, wherein a freezing period is 1 day or more.
  7. The method of Claim 1 or 2, wherein a freezing temperature is -20°C or lower.
  8. The method of Claim 1 or 2, wherein the removing of the insolubilized protein from the extract is performed by centrifugation or filtration.
  9. The method of Claim 1, further comprising a step of removing insoluble substances from the plant-derived extract before the step (1).
  10. The method of Claim 9, wherein the removing of insoluble substances from the plant-derived extract is performed by centrifugation or filtration.

Description

Technical Field The present invention relates to a method for removing a plant-derived soluble protein from a plant-derived extract, and the like. Background Art Technology for producing substances using organisms such as plants, animals, or microorganisms is capable of material production from biomass, which does not depend on fossil resources. Therefore, it is anticipated to lead to the realization of a carbon-circulating society and manufacturing innovation toward sustainable economic growth. Among these, substance production by plants using genetic recombination technology has the potential to express proteins that are difficult to produce due to complex structures or high molecular weights, and is seen as promising for utilization in fields such as pharmaceuticals, food, and healthcare. However, there is still little commercial track record, and technologies for mass production have not been established. In particular, the separation and purification of the target expressed protein requires multiple reagents and multi-step operations (Patent Literature 1), and there is a great demand for an efficient purification method. In particular, there is a great demand for a purification method that can efficiently remove a plant-derived soluble protein, such as RuBisCO, which are contained in large quantities in plant-derived extracts. Citation List Patent Literature Patent Literature 1: JP 6560495 B Summary of Invention Technical Problem An object of the present invention is to provide a method for removing a plant-derived soluble protein, which is present in large quantities, from a plant-derived extract. Solution to Problem The present inventors centrifuged crude extracts of wild-type Nicotiana benthamiana and N. benthamiana into which a green fluorescent protein (GFP) gene had been introduced, separating them into a supernatant (S1) and a precipitate (P1). Furthermore, S1 was subjected to freeze-thaw treatment, centrifuged, and separated into a supernatant (S2) and a precipitate (P2). Subsequently, the soluble proteins contained in S1 and S2 were separated by SDS-PAGE, and the polyacrylamide gel was stained with Coomassie Blue. As a result, it was confirmed that Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a plant-derived soluble protein confirmed to be present in large quantities in S1, had almost disappeared in S2. At the same time, by subjecting S1, from which the precipitate P1 had already been removed by centrifugation, to freeze-thaw treatment and centrifugation, a new precipitate P2 was formed, which was considered to be a new insoluble fraction containing RuBisCO. Furthermore, analysis of the amount of GFP contained in S1 and S2 by Western blotting revealed that the freeze-thaw treatment did not affect the solubility of GFP, which remained in the supernatant. In addition, the inventors found that even a short freezing treatment of about 10 minutes could remove some plant-derived soluble proteins, and a 60-minute freezing treatment could remove a sufficient amount of RuBisCO; the present invention could be implemented even when using GUS protein or human FGF1 as the exogenous protein; and that many types of plant-derived soluble proteins other than RuBisCO could be removed by the freeze-thaw treatment. From the above, it became clear that the freeze-thaw treatment insolubilizes plant-derived soluble proteins, including RuBisCO, while not affecting the solubility of exogenous proteins such as GFP, GUS, or FGF1, consequently enabling the separation of both. Furthermore, the freezing time did not affect the above-mentioned effects. The present inventors conducted further research based on these findings and completed the present invention. That is, the present invention is as follows. [1] A method for removing a plant-derived soluble protein from a plant-derived extract, the method comprising the following steps: (1) a step for insolubilizing the plant-derived soluble protein by freezing and thawing the plant-derived extract, and(2) a step for removing the insolubilized protein from the extract.[2] The method of [1], wherein the plant-derived soluble protein comprises RuBisCO.[3] The method of [1] or [2], wherein the plant is a plant belonging to the genus Nicotiana.[4] The method of [3], wherein the plant belonging to the genus Nicotiana is Nicotiana benthamiana.[5] The method of any one of [1] to [4], wherein the plant expresses an exogenous soluble protein which is not insolubilized by freezing and thawing.[6] The method of any one of [1] to [5], wherein a freezing period is 1 day or more.[7] The method of any one of [1] to [6], wherein a freezing temperature is -20°C or lower.[8] The method of any one of [1] to [7], wherein the removing of the insolubilized protein from the extract is performed by centrifugation or filtration.[9] The method of any one of [1] to [8], further comprising a step of removing insoluble substances from the plant-derived extract before the step (1).[10] The me