JPH0977789A - Solubilization of insoluble protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solubilize an insoluble protein manifested in a recombinant host. SOLUTION: An insoluble protein manifested in a bacterium is solubilized by treating the insoluble protein with a solution having higher pH than the pH of a culturing medium in the bacterium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for solubilizing insoluble proteins. More specifically, the present invention relates to a method for activating an insoluble protein produced when a heterologous protein is expressed in bacteria.

[0002]

2. Description of the Related Art In the field of gene recombination, a bacterial expression system is used as a means for producing a target protein in a large amount and easily recovering it. In particular, when the expression system of E. coli is used, a large amount of the desired product (protein) is accumulated.

However, when a heterologous protein is expressed in bacteria, it is often recovered as a granular insoluble protein such as an inclusion body.

Therefore, a method for solubilizing this insoluble protein is desired. The solubilization activates proteins, especially enzymes and hormones. As one method of solubilization, for example, when the insoluble protein is a thermostable enzyme, a method of solubilizing the enzyme by heat treatment and simultaneously activating the enzyme is known.

However, this method cannot be used for other than thermostable enzymes. Therefore, a new activation method has been desired.

[0006]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a method for solubilizing insoluble proteins.

[0007]

The present invention is a method for solubilizing an insoluble protein produced when a heterologous protein is expressed in bacteria, the method including treatment with a solution having a high pH.

In a preferred embodiment, the bacterium is
E. coli.

In a preferred embodiment, the method comprises:
Including heat treatment.

[0010] In a preferred embodiment, the protein is a thermostable enzyme.

[0011] In a preferred embodiment, the thermostable enzyme is a thermostable amylase.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention can be applied to any protein as long as it is an insoluble protein produced when a heterologous protein is expressed in bacteria.

In the present invention, the bacterium means a so-called prokaryote, and is a concept including both gram-positive and gram-negative bacteria.

The method for expressing and producing a heterologous protein using bacteria as a host is well known to those skilled in the art. For example, a plasmid in which a gene encoding a heterologous protein of interest is operably linked to a vector replicable in a bacterial host may be used.

Those skilled in the art are well aware of a vector capable of replicating in a bacterial host, a method of ligating a gene of interest to the vector to obtain a plasmid, transformation of bacteria using this plasmid, and selection of a transformant strain. . The method for culturing the obtained transformant is also well known to those skilled in the art.

Heterologous proteins expressed in bacteria generally form so-called inclusion bodies and become insoluble. In the present specification, it is said that an insoluble protein is produced when a granular substance is observed by observation with a microscope.

The insoluble protein can be obtained by a method well known to those skilled in the art, for example, in the case of Escherichia coli, by a cell disruption treatment such as osmotic pressure treatment or ultrasonic treatment, in the medium or in a buffer solution having a pH substantially equal to that of the medium. It can be recovered by disrupting the cell wall in and centrifuging.

The present invention includes treating with a high pH solution. High pH usually refers to a pH of 6 or higher.
The optimum pH varies depending on the secreted protein, but preferably 2 to 6, more than the isoelectric point of the target protein,
Preferably, a pH of 3 to 4 higher can be used. pH
The treatment time is usually about 1 minute to 20 minutes, preferably about 3 minutes to 10 minutes, but it may vary depending on the treatment temperature.

In a preferred embodiment, the method comprises
Including heat treatment. This method is particularly effective when the protein used has heat resistance. When it does not have heat resistance, the heat treatment time is shortened to prevent heat-induced deactivation. Here, heat resistance means 65 in the present specification.
It refers to the case where it has a residual activity of 50% when heat-treated at 10 ° C. for 10 minutes. The heat treatment can shorten the pH treatment time.

The degree of solubilization can be determined by measuring the activity of the protein. That is, it can be determined by measuring the activity of the protein contained in the pH-treated solution, for example, the enzyme activity. By measuring the specific activity, the extent of solubilization can be determined.

[0021]

EXAMPLES Hereinafter, the case of using a thermostable KOD1 acid amylase will be described as an example, but it goes without saying that the present invention is not limited to such an example. (Preparation of KOD1 acid amylase expression plasmid) E.co
liHMS174 (DE3) pLysS (pEDS-3) was prepared as follows.

Chromosomal DNA derived from the Pyrococcus KOD1 strain was purified by a conventional method and cut with EcoRI. This EcoRI fragment was ligated into the EcoRI site of commercially available pUC18. E. coli HS174 was transformed with the obtained plasmid, and an amylase-producing strain was selected. The resulting plasmid was named pNMT-16. FIG.
The restriction enzyme map is shown in.

The EcoRI fragment of pNMT-16 (7.5 kb) was cut out,
The base sequence was determined. Determine the coding region (about 1.3kb)
It was amplified by PCR and cloned into the NcoI-BamHI site of pET8C vector to prepare plasmid pEDS-3. The fabrication is shown in FIG.

(Recovery of insoluble protein) The plasmid pEDS-3 obtained above was transformed into E. coli HMS174 according to a standard method. The transformant selected with 50 μg / ml of ampicillin was cultured in 2.5 L of LB medium at 37 ° C. and OD
When 660 = 0.4, IPTG was added to a final concentration of 1 mM. It was further cultured for 4 hours. When the culture solution was observed under a microscope, E. coli cells were found to be granular. The culture solution was centrifuged (8,000 rpm, 10 minutes) to collect the cells. The collected E. coli was treated with 50 mM sodium acetate buffer (pH 5.
5) Suspended. The suspension was sonicated at 0 ° C. for 20 seconds and the interval of 1 minute was repeated 7 times to disrupt the cells.
The disrupted liquid was centrifuged (12,000 rpm, 20 minutes) to obtain an insoluble amylase fraction. FIG. 3 shows the results when the soluble fraction as a supernatant and the insoluble fraction as a precipitate were stained with CBB (Coomassie Brilliant Blue) and with active staining, respectively. The activity staining was performed by Lee et al., Appl.Environ.Mi.
crobiol. 60, p3746-3773.

(Solubilization of insoluble fraction) The protein amount corresponding to 25 ml of the culture solution of the obtained insoluble amylase suspension was adjusted to 50 mM.
Sodium acetate buffer (pH 5.5), and 50 mM Tris-
Suspend in 1 ml of Hcl buffer (pH 7.0 and 8.5) at 37 ℃
Processed in. At the same time, heat treatment (60 ° C and 85 ° C) was performed.
The reaction time was sampled every 0, 10, 30 and 60 minutes, and centrifugation (14,000 rpm, 10 minutes) was performed to collect the supernatant, and the amylase activity and the amount of solubilized protein were measured to determine the specific activity. The results are shown in Table 1.

[0026]

[Table 1]

At pH 5.5, which is close to the pH of the medium, almost no solubilization occurred at 37 ° C, but at pH 7 or pH
It can be seen that the treatment of 8.5 solubilizes and restores the activity. Although the effect of heating is also recognized, the effect of pH is greater.

FIG. 4 shows SDS-PAGE of the supernatant fraction after the treatment. It can be seen that the amount of solubilized protein increases as the pH and temperature rise.

(Recovery of solubilized protein) 50 mM Tris-Hcl (p
When the amylase was suspended in H7.0) and heat-treated (85 ° C, 10 minutes) to be solubilized, the amylase was subjected to hydrophobic chromatography using Phenyl Superrose, and the amylase was adsorbed on the carrier. After washing the column with 50 mM Tris-Hcl (pH 7.0), amylase was eluted with 1 mM Tris-Hcl (pH 7.0). The eluted amylase showed a single band on SDS-PAGE. (Figure 5, lane 5)

[0030]

INDUSTRIAL APPLICABILITY The insoluble protein expressed in bacteria is easily solubilized by treating it at a pH higher than that of the medium. Therefore, according to the method of the present invention, the insoluble protein obtained by the recombinant method can be easily solubilized and recovered in an active form.

[Brief description of drawings]

FIG. 1 shows the plasmid pNMT-16.

FIG. 2 is a diagram showing the construction of plasmid pEDS-3.

FIG. 3 is a photograph of an electrophoretic image showing the distribution of proteins in the supernatant and the precipitate after cell disruption.

FIG. 4 is a photograph of an electrophoretic image showing the degree of solubilization when the insoluble fraction was treated at various pH and temperatures.

FIG. 5 is a photograph of an SDS-polyacrylamide electrophoresis image of amylase in each fraction.

Claims (5)

[Claims] 1. A method for solubilizing an insoluble protein produced when a heterologous protein is expressed in bacteria, which comprises treating with a solution having a high pH. 2. The method according to claim 1, wherein the bacterium is Escherichia coli. 3. The method according to claim 1, further comprising heat treatment. 4. The method according to claim 1, wherein the insoluble protein is a thermostable enzyme. 5. The method according to claim 4, wherein the thermostable enzyme is a thermostable amylase.