JPH01233299A - Solubilization of insoluble protein - Google Patents

Abstract

PURPOSE: To easily and rapidly solubilize the insoluble protein in a sufficient yield, without requiring any modifier by allowing insoluble protein expressed by cultivated cells (preferably, Escherichia coli) to contact with an ion exchanger and separating the treated protein from the ion exchanger.

CONSTITUTION: This method comprises: allowing insoluble protein expressed by cultivated cells (preferably, Escherichia coli) to contact with an ion exchanger; and then, separating the treated protein from the ion exchanger to solubilize the protein. At this time, the contact of insoluble protein with an ion exchanger comprises: allowing the protein to contact with the ion exchanger in the presence of finely-divided cells or cell fragments in a culture solution; and further, preferably using as the ion exchanger, a spherical anion exchanger such as Q-Sepharose(R) or spherical cation exchanger such as S- Sepharose(R).

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for stabilizing insoluble proteins expressed from cultured cells.

Cultured cells, especially bacteria (especially Escher
ichia colt) is used industrially for the overproduction of proteins, especially those encoded by divergent genes in different organs. In many cases, overexpression of proteins of commercial interest can be carried out satisfactorily (as illustrated by the examples of β-interferon and insulin). It turns out that there is a problem with E, C.
For o11, see Biotechnology, 5
(1987) 883-890. Proteins precipitate as visible inclusions under electron microscopy (e.g., 5science 215 (1982)
687-689). The above inclusions offer the advantage that the recombined protein is well protected before proteolytic degradation (e.g. EMBO Journal 3 (198
4) See 1429-1434). However, it is extremely difficult to separate the soluble or active form of the recombined protein from the inclusions or precipitates. Often, urea, sodium dodenolsulfate (SDS) or another denaturing agent is used to solubilize proteins. However, this type of purification results in undesirable denaturation of the protein. For example, see the report below. Mar
s ton (1987), “The Puri
fication of enkaryotic
polypeptidesexpreSsed in
Escherichia coli, ”, ON
A Cloning.

Vol, Ill, ed, D, Glover+
p. 59-88 IRL Press.

0xfordHPNAS, 80 (1983) 906
~910 or PNAS.

82 (1985) 2354-2358° However, reconstitution involves a loss of active material. This is because reconstruction is generally not complete. In some cases, renaturation is not possible and therefore only the soluble fraction can be recovered and the insoluble protein must be discarded. For example, B.
iotechnology, 5 (1987) 960
See ~965.

Now, the purpose of solubilizing insoluble proteins extracted from cultured cells is achieved, based on the present invention, by a method in which the insoluble proteins are brought into contact with an ion exchanger and then separated from the ion exchanger.

The essential advantage of the strategy according to the invention is that no denaturing agents are required, the method according to the invention is easy and rapid to carry out, and the solubilized protein is obtained in almost satisfactory yields.

It is known that after centrifugation of the insoluble material, proteins are adsorbed onto ion exchangers; in this known method, of course, only soluble proteins are used.

For example - Current ProLocoIs in
Molecular Biology.

198L p-10,9,2+ N, Y, (Gre
ene Publ, Ass, and Wice
y Inter-5science). This prior art is
It only makes sense if an appropriate ion exchanger can be selected for the particular protein used.

Industrially cultivated bacteria (especially E, coli)
It can be analogized that the method of the present invention can be applied to proteins extracted from. However, the method according to the invention can also be applied to proteins expressed from industrially cultured eukaryotic cells (e.g. insect cells).
, 84 (1987) 5700-5804 or Ge
netic Engineering, 8 (1986
) 27? See -298.

When carrying out the method according to the invention, insoluble proteins can be brought into contact with an ion exchanger in the presence of disaggregated cells and cell debris in a culture medium. Of course, after separating the disintegrated cells and cell debris, the insoluble proteins can be contacted with an ion exchanger.

Routine suitability experiments can be performed when selecting the appropriate ion exchanger for a particular protein.

Furthermore, the prior art mentioned above helps with orientation;
The ion exchanger may be an organic matrix or an organic polymer or polysaccharide (e.g. agarose).
An example of an ion exchanger that can be used according to the invention is Sepharose. A special example of an ion exchanger that can be used universally for protein solubilization is Q-Sepharose (
Pharmacia's Q-3epharose-Fas
t-flow's Q-Sepharose is an example of a cation exchanger. Cation exchangers of this type are used in the case of basic media. However, in the case of acidic media, anion exchangers can also be used, as an example:
S-Sepharose (Pharmaeia's S-5-5e
pharose-Fast-Flo.

For overproduction of particular proteins, for example, when using bacterial cells, the cells can be lysed in a known manner, for example by ultrasound, lysozyme, osmotic shock, freezing/thawing or a combination of the above strategies.

For the protection of proteins, it is preferable to use a buffered medium; when working on a large scale, it is advantageous to add ion exchangers to the buffered culture medium without separating the disintegrated cells and cell debris. be. If a particulate ion exchanger is used, the ion exchanger can be filtered or centrifuged from the culture medium, for example after 1-2 hr. In some cases, for example by adding a salt-containing buffer,
The separation of the ion exchanger from the protein-containing liquid medium, in which the adsorbed proteins have to be separated from the ion exchanger, can likewise be carried out by filtration or centrifugation. The protein-containing liquid medium can be processed in known manner for protein recovery.

The invention will now be described in detail with reference to eight exemplary embodiments and a drawing.

1JIJL top: Solubilization of 5V4Q-T-antigen derivative Th (peptide Th) 5 ml of overnight cultured E, col+ cells (JMI O3) containing plasmid pTh were treated with isopropyl-β in 100 ml of Shiichi broth medium at 37°C. -D-Thiogalactopyranoside (IPTG) 5ml induced for 4 hours.

Then, a centrifuge (Sorvall Rotor 5
s34) and centrifuged for 5 wins at 3.0OOrpn+. The supernatant, i.e. L-pross medium, was drained and the pellet was suspended in buffer A 2.5+1 (buffer A: Tris-Hcl (pH 8,0) 50 mM,
NaCl 50mM, E[lTA 1mM,
RoTT 1mM.

PMSF 1mM and glycerin 1O%), lysozyme (5μ/m
l) was added, the suspension was kept on ice for 5ml, irradiated with ultrasonic waves for 3 x 20sec, and placed on ice again for 15ml.
n+in was retained. The lysate was added to the same volume of Q-Sepharose Fast Flow and equilibrated in buffer.

The system was then shaken for 2 hr at 4° C. on a raw data and then centrifuged at 5.000 rpm for 15 mjn. The supernatant was discarded. Add the precipitate to the same volume of buffer I.
B (Buffer B: Buffer A with 250 mM NaCl) was then shaken for 15 Ilin at 4 °C on a raw data, then 1 at 5,000 rpm.
Centrifuged for 5+ min. The supernatant was used to measure Th activity in a DNA binding test. The results are shown in Table 1.

Table 1 DNA binding test component of Th (N based on Example 1) Volume Densitometer Th1M degree [INA binding (μg) measurement (a (μg) activity □ (%) (%) Total lysate 500 5 .6 2 100
Supernatant liquid of Roseph Arrows binding process 500 0.0 0 00
- Eluent of solution from Sepharose 500 16.7 0 298
Solubilization of SV40-T-antigen (Th) The procedure was carried out in the same manner as in Example 1. However, in this example, after cell dissolution, the cells were centrifuged at 4° C. for 15 ml, and the supernatant and the precipitate resuspended in buffer A were treated with an ion exchanger. The activity of the restored protein was confirmed in the same manner as in Example 1 (see Table 2).

Table 2: DNA binding test component of Th (separated according to Example 2) Volume Densitometer rhtW degree [IN^Binding (μl) Measured value (μg) Activity □□ (%) (%) Total lysis 1 500 5 .6 2 100
Q-Sepharose binding process supernatant 500 5.8 2 99Q
- Eluent of lysate from Sepharose 500 10.9 4 194
JJIJL↓: Solubilization of interleukin-2 (TL-2) The same procedure as in Example 1 was performed. However, in the case of this example,
Plasmid ptac4 instead of 5V40-T-antigen
TL-2 prepared by E. coli cells was solubilized. Unlike Example 1, NaCl in Buffer B
The concentration was increased to 500mM. The activity of solubilized rL-2 was confirmed by T cell growth test (see Table 3).

Table 3: (CI-3-Activity test of IL-2 by T-cells (Separate I1--12 based on Example 3) Component TI of thymidine, activity of -2-G 11 - Rule Aj]) (9i
Yu=-goo-total solution 2617.0
1000-Sepharose-FF Supernatant liquid of the binding process 178.0 7 volumes of dissolved solution from 70-Sepharose-FF 5379.5 205 type can-9 soil: Interleukin 2 (TL-2) The procedure was carried out in the same manner as in Solubilization Example 3. However, in the case of this example,
After cell dissolution, the cells were centrifuged at 15 mL at 4° C., and the supernatant and precipitate resuspended in buffer were treated with an ion exchanger. The activity of the solubilized protein was confirmed in the same manner as in Example 3 (see Table 4). activity of -------m--Nobuaki'b engineering)-(%) -Total lysate 2617.0 1000-Sepharose-FF Supernatant of binding process 2583.0 99Q Sepharose-FF Supernatant eluent 2808.5 107 Solubilization of Jliv-myb by E. coli HBIOI (under TRP monitoring;
pvm2028) produced oncoprotein sea Nvb
The inclusion body of S-Sepharose-Fast-Flow (S
-Sepharose-FF). In this case, no anion exchanger adsorption was observed.

Solubilization of T260 In the same manner as in Example 5, the T-antigen-peptide 206 inclusion complex was solubilized with S-Sepharose.

Similar to Example 1, membrane protein inclusions, which form the basis of the resistance of the new type of rhifampicin, were added to Q-Sepharose and phenylSepharose. Solubilized with

IPT' G-isopropyl-β-D-thiolactopyranoside E D T8-ethylenediaminetetraacetic acid DTT = dithioerydrift PMS F-phenylmethylsulfonyl fluoride c p
m = counts pro win material EI IJ
-Top left E, coli JM103 Nucleic Ac1
ds Res, 9 (1981) 309-321 Reference E, coli HBIOI J, Mo1. Biol,
, 4 (1969) 459-472 CL3-T (cell) Bioehea+1stry, 22 (198
3) Reference 251-255 PVM2028 (Boo, Sumido) EMBOJ,
, 6 (1987) 2719-2725 Reference PTh (Plasmid) J, Virol, , 62
(1988) 1999-2006 reference TRP is a regular sales promoter.

Claims (1)

[Scope of Claims] 1) A method for solubilizing insoluble proteins expressed from cultured cells, which comprises bringing the insoluble proteins into contact with an ion exchanger and then separating them from the ion exchanger. 2)E. The method according to claim 1, characterized in that the protein expressed from Coli is solubilized. 3) The method according to claim 1 or 2, characterized in that the protein is brought into contact with an ion exchanger in the presence of disintegrated cells and cell debris in a culture medium. 4) The method according to claim 1 or 2, wherein the protein is brought into contact with an ion exchanger after separating the disintegrated cells and cell debris. 5) Process according to one of claims 1 to 4, characterized in that a spherical ion exchanger is used. 6) Claims 1 to 3, characterized in that an anion exchanger (especially Q-Sepharose) is used as the ion exchanger.
A method according to one of clauses 5. 7) Claims 1 to 3, characterized in that a cation exchanger (especially S-Sepharose) is used as the ion exchanger.
A method according to one of clauses 6. 8) Method according to one of claims 1 to 7, characterized in that the protein is separated from the ion exchanger by elution.