TWI311562B - One step rapid desalting and protein refolding process - Google Patents

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel single-step protein desalination and folding method for protein purification, which is different from the conventional chromatography column method. After the protein is purified in large quantities under denaturing conditions, 'by removing the denatured salt under the protection of highly hydrophobic molecules through the protein concentrating column or protein ultrafiltration membrane, and keeping the protein under denaturing conditions during the salt removal process' The process of high ^ hydrophobic molecules to control the refolding of proteins. [Prior Art] In the post-genome era, proteomics began to rise, and research on protein structure and function has received much attention. At present, many analytical methods for protein structure have been carried out in various countries, and many important target proteins need to be expressed in large quantities and obtain physiologically active proteins (see, S. Yokoyama, Cwrr. Opin. Chem. Biol 2003; 7(1), 39-43 ) ° ^ ^ The biotechnology pharmaceutical industry is highly developed today, and many recombinant protein ❿ drugs are in increasing demand. Due to the maturity, easy operation and low cost of the prokaryotic expression system, many proteins are currently available. It is obtained in large quantities by the prokaryotic performance system. However, 'the prokaryotic expression system can be used to obtain proteins quickly and in large quantities,' but often the protein after its expression is an inclusion body, because prokaryotic cells are not as hot as eukaryotic systems. Heat shock protein or Chaperon to assist in protein folding. In order to solve this shortcoming in the laboratory or the short workmanship, it is often used to first utilize the protein denaturation method\deI;

5 TBH03-P060009-TW 1311562 condition) Denature the protein to achieve solubility. The normal physiological configuration and activity of the protein is then re-assigned by removing the denaturing conditions. Traditionally, Pr去tein desalting and refolding can be performed in several ways: 1. slow dialysis method, 2. dilution method, or 3. small molecular sieve The amount of desalting column is achieved (see, for example, Natalia et al., On column chemical refolding of protein Pharmagenomics (2004) pp. 22-26). However, the above several methods each have their disadvantages. For example, for the slow dialysis method, if the target protein has an unstable property, protein degradation is caused because the slow dialysis method requires long-time dialysis. "Dilution method can cause insoluble aggregation for proteins with a large number of Hydrophobic domains; or protein degradation due to rapid changes in ion concentration for salt sensitively." (protein degradation) ° • Therefore, for less stable and salt-sensitive proteins, column chromatography is often used for desalting and refolding. For the promotion of the amount of protein, if the tube column chromatography method is used to directly replace the larger column, a high amount of elution buffer is required, and the longer column also represents more. Long operating time. However, the most important problem with the 矣 salt column in process amplification is the column of twice the enlarged volume, which is not equivalent to twice the protein purification capacity. For some proteins that must be maintained in the denatured state of the protein, the rapid desalting of the desalted column causes the protein to be sensitive to ionic strength and the protein

6 TBH03-P060009-TW 1311562 Degradation of the quality during desalting. To this end, it is necessary to develop a number of methods of operation that maintain the protein in a manner close to the denatured configuration. & Previous literature reports (D. R0zema and SH GeUm 35(49), 1 5760-1 577 1 ), a water-based substance model that simulates nuclear protective proteins to prevent insoluble deposition during protein denaturation. A method in which a water-based substance is used to fold a protein, such a t ^ 濩 濩 蛋白 蛋白 ( ( ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar ar 。 。 。 。 This type of semi-successfully allows the protein to go to the salt column without the aid of a chromatography column, but it still takes a long time to carry out, and the protein is lost during the filtration process. Therefore, this: The artificial defensive protein unfolding method described in the essay, the development of the protein concentrating column (centric 〇n) or protein super cel.l) for rapid desalting and protein refolding = method. s ir [Summary] The purpose and advantages will be partially described below. The objective is to provide a novel single-step method which is characterized by the desired protein = after, by means of a protein concentrating tube south of a hydrophobic salt (such as urea), and under the salt-removing conditions Refolding by removal of highly processed proteins. The invention has the characteristics of the present invention. (1) The invention will be described in the description of the invention by extensive purification of the containing or cleavable protein to change the hydrophobic protein in the column or egg molecule. Desalting and folding of the square conditions, a large number of pure white super-membrane, under the protection of the process of removing the protein from the degeneration, control the salt and folding method according to the present invention

TBH03-P060009-TW 7 1311562 Protein denaturation solution is mixed with a solution containing highly hydrophobic molecules; (2) the mixture is passed through a protein concentrating column or protein ultrafiltration membrane; and (3) desalted by step (2) The protein solution removes the highly hydrophobic molecule by progressive centrifugal displacement, and the denatured protein is refolded into an active molecule. As used herein, "denatured protein" refers to a protein that requires extensive purification in a denatured manner due to its insolubility. As used herein, "highly hydrophobic molecule" refers to a hydrophobic material that is used in the process of altering protein denaturation (removing denaturing salts) to prevent insoluble deposition of the desired protein, as is known in the art, and Examples of hydrophobic molecules that can be utilized in the present invention include, but are not limited to, hydrophobic molecules as described in Natalia et al., "On column chemical refolding of protein Pharmagenomics (2004) pp. 22-26n, however, any Hydrophobic compounds such as Triton X-100, tween_20 or polyphenylene ring compounds which can be used can be applied to the method of the present invention. According to the present invention, progressive centrifugation replaces the condition of highly hydrophobic fractions, and the proteins are separated as desired. In a preferred embodiment of the invention, the denatured HOXB4H protein is subjected to a centrifugal speed of about 1000 to about 2500 g/min, and is successively replaced by 5 to 10 centrifugation to obtain a low to high concentration. a solution of a macromolecular hydrophobic substance such as cyclodextrin, which removes the high-k water molecule, and refolds the denatured HOXB4H protein into a natural form. (Native form) ° [Embodiment

8 TBH03-P060009-TW 1311562 The present invention will describe in detail specific embodiments. These specific examples are provided by way of illustration of the invention and are not intended to limit the invention. The present invention is intended to embrace such modifications and alternatives and modifications. Example 1. Desalting of denatured HOXB4H protein by 10K centricon The denatured protein used in this example was φ H0XB4H, a method for which it was extensively manufactured and purified, as described in the related application 094,122,467. Add a new 1 OK centricon (50 ml or 1 5 ml) concentrator to (1 〇ml or 3 ml) of secondary water and centrifuge 3 rpm, l〇mins to remove the upper and lower secondary water. Pre-processing. Mix high-salt samples containing denatured HOXB4H protein with 1:4 (protein solution 1, D-PBS-T 4 mixed) with D-PBS-T (double phosphate buffer solution containing 0 1% Triton X-100) Add the resulting mixture to the pre-treated concentrate tube and centrifuge 3000 rpm '1 〇mins to replace the protein denatured salt Buffer to d-PBS-T buffer at 80% rate to bind HOXB4H The hydrophobic (Hydrophobic) region of the protein. Then, the desalted sample buffer was sequentially replaced with a solution containing i mM, 2 mM, 3 niM, 4 mM, and 5 mM β-cyclodextrin (beta-cyclodextrin) in the presence of D-PBS buffer at a centrifuge rate of 1000 to 2500 g/min. In which the centrifugation was performed twice at each concentration, and then the buffer was replaced with D-PBS. The protein solution was centrifuged at each centrifugation rate, by using the concentration (30 ° / 〇 BSA plus 90%). Glycerol) at 1:5

TBH03-P060009-TW 9 .1311562 (protein solution) mixed with the protein solution, 3 white / 5% said to add 15% - two deposits in the second: / water phase. Human Example 2. Evaluation of the refolding of the purified H0XB4H protein, and evaluation of the HOXB4H protein by the protein desalting method of the present invention, which can be refolded into the nucleus. The protein is transported into the cell by TAT, which is carried. The protein 'must be in a denatured state or close to a denatured state to facilitate the egg to enter the cell. Because the TAT medium enters the way, it is not like the traditional protein entry mode. It is entered by energy or a specific channel. The TAT media entry is the first one. When the positively charged tat is in contact with a negatively charged cell mill, the protein that is carried by the protein is crossed in a straight line through the cell membrane. The protein that is over-folded cannot be unblocked and becomes incapable of entering the cell (see, TATSteven R.

Schwarze, Keith A. Hruska and Steven F. Dowdy,

Trends in cell biology 10,290-295 ) > Differently, different centrifugal G values (500g, 750g, l〇〇〇g, l25〇g, 1500g, 1750g, 2000g, 2250g and 2500g) were carried out as in the example The protein sample obtained by the PBS-T replacement described above was used to detect whether the HOXB4H protein protein has the ability to enter the nucleus by Western blotting. Twenty-three T cells were cultured in a 10 cm culture dish to reach 1 〇6 cells, and TAT-HOXB4 was added to the culture dish at a final concentration of 15 nM under various centrifugation conditions as described above. Rinse with D-PBS

After centrifugation (1000 g for 5 min), the protein was extracted with RIPA (Radio Immunoprecipitation Assay buffer; 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% NP-40, 0.1% SDS, 1 mM PMSF), followed by electrophoresis and then collapsed. To NC

10 TBH03-P060009-TW *1311562 On the membrane, the presence of ΗΟχΒ4Η protein was detected by Western blotting. The primary antibody used was an anti-His tag antibody (purchased from Sigma), and the secondary antibody was an anti-mouseigG antibody (purchased from Sigma), and both were allowed to act at 37 ° C for 2 hours. The results are shown in Fig. 1 'where C is a 293T reference cell' as a negative control negative control group, and p is a greatly purified denatured protein as a positive control group. From the results, it was found that the denatured HOXB4H protein can be imparted by the method under appropriate conditions.

[Simple description of the figure] The first picture shows the results of using Western blotting to detect whether H0XB4H protein enters the nucleus. Wherein C is a 293T reference cell; Lanes 1 to 9 represent 1 mM, 2 mM, 3 mM, 4 mM at a centrifuge rate of 500 g, 750 g, 1000 g, 1 250 g > 1 500 g '1 750 g, 2000 g, 2250 g, and 25 00 g, respectively. And 5 mM β-cyclodextrin solution was subjected to PBS-T replacement, and centrifuged twice at each concentration; Lane 10 showed that it was centrifuged only 10 times with 5 mM β-cyclodextrin solution at a centrifuge rate of 2000 g; It is a Η4Η denatured protein after extensive purification.

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Claims (1)

Ϊ311562 0 (Μ修浼) i , the scope of patent application: = ^ method for the purification and refolding of the denatured protein purified by the affinity column, comprising: 匕 1 containing a large number of purified and affinity column a solution of the desired denatured protein under the variation and a solution containing a highly hydrophobic molecule selected from Tm〇n χ100 or tween_20 are mixed in a mixing ratio of 1:3 to 1:5; the mixture is passed through a protein concentrating column Or σ protein super-film is centrifuged at a centrifugal speed of 1000 to 3000 g / min for 5 _ 10 minutes for rapid desalting; and (c) step (b) desalted protein solution at 1 〇〇〇 2 5 离心g/min at a centrifugation speed for 5-10 centrifugation to remove the two-degree hydrophobic molecule to refold the s-denatured protein, wherein the progressive centrifugation is performed from a low concentration to a high concentration of β The cyclodextrin (β-cyclodextrin) solution sequentially displaces the highly hydrophobic molecule. 2. The method of claim 1, wherein the denatured protein is a ΗΟΧΒ4Η denatured protein. 3. The method of claim 1, wherein the progressive centrifugation is performed by sequentially replacing the desalted sample buffer with ImM, 2 mM, 3 mM at a centrifugation speed of 1000 to 2500 g/min. A solution of 4 mM and β-cyclodextrin in D-PBS buffer was present. 12 TBH03-P060009-TW