JP2003508054A - Method for producing channel-forming protein - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for producing a channel-forming protein found in Gram-positive bacteria, wherein the channel-forming protein is obtained by: a) heterologous overexpression, or b) purification from mycobacteria, wherein the extraction temperature is For processes above 50 ° C.

Description

Detailed Description of the Invention

The present invention relates to a method for producing a channel-forming protein, a channel-forming protein, a gene encoding such a protein, and mspA and mspC.
Or mspD gene, plasmid vector, and overexpression system.

The present invention relates generally to the technical field of manufacturing nanostructures. To date, the best characterized nanostructures are carbon nanochannels (Yakobson, BI and Smalley,
RE Fullerene nanotubes: C _1,000,000 and beyond. "Am Sci" 85, 324, 1997)
. It has been shown that the electronic properties of carbon nanochannels can be controlled by their structural details. Carbon nanochannels are manufactured by CVD (Chemical Vapor Deposition) method (
Fan, S., Chapline, MG, Franklin, NR, Tombler, TW, Cassell, AM an
d Dai, H. Self-oriented regular arrays of carbon nanotubes and their fie
ld emission properties. “Science” 283, 512-4, 1999), which was synthesized using different variants and is therefore very expensive.

Johnson, SA, Ollivier, PJ and Mallouk, TE “Ordered mesoporous pol
ymers of tuneable pore size from colloidal silica templates. ”,` `Scienc
e ”283, 963-965 (1999), a technique for producing organic nanochannels based on a template is reported. This method can be used to generate nanochannels with diameters of 5-35 nm.

[0004] Mycobacteria belong to a subgroup of Gram-positive bacteria, which subgroup contains mycolic acids, examples of which are Corynebacterium, Nocardia, Rhodococcus, Gordona, Tucumella, and Dietia. Genus. Trias, J. and Benz, R. `` Permeability of the cell wall of Mycobacterium
smegmatis. ”“ Mol Microbiol ”, 14, 283-290 (1994) describes a channel-forming protein called porin in the mycolic acid layer of mycobacteria. No biochemical or molecular genetic data for these porins have been published.

Lichtinger, T., Burkovski, A., Niederweis, M., Kramer, R. and Benz, R.
`` Biochemical and biophysical characterization of the cell wall porin of
Corynebacterium glutamicum: the channel is formed by a low molecular ma
ss polypeptide. ”,“ Biochemistry ”37, 15024-32 (1998), a method for preparing porin from corynebacteria is known. This technique is relatively inefficient.

[0006] Mukhopadhyay, S., Basu, D. and Chakrabarti, P. "Characterization of a
porin from Mycobacterium smegmatis. "," J Bacteriol "179, 6205-6207 (199
7) describes the extraction of porin from M. smegmatis by incubation with a buffer containing 1% Zwittergent for 1 hour at room temperature. The yield was poor and the porin was contaminated with many other proteins.

Harth, G. et al., “High-level heterologous expression and secretion in rapid
ly growing nonpathogenic mycobacterium of four major Mycobacterium tuber
culosis extracellular proteins considered to be leading vaccine candidat
es and drug tatgets. ”,“ Infection and Immunity ”65, 2321-2328 (1997), it is known that strong expression of a mycobacteria-specific protein in Escherichia coli is not considered possible.

Senaratne, RH et al. “Expression of a Gene for a Porin-Like Protein of th
e OmoA Family from Mycobacterium tuberculosis H37Rv. ”,“ J Bacteriol ”1
80, 3541-3547 (1998) in Mycobacterium tuberculosis (M.
The expression of a gene for a porin-like protein from ycobacterium tuberculosis) H37Rv is described. Expression of the gene results in a discontinuity in bacterial growth, apparently because the expressed protein is toxic to E. coli. A very small amount of protein from E. coli could be isolated just before cell death.

It is an object of the present invention to provide an improved process for the production of channel forming proteins. This object is defined in claims 1, 26, 27, 28, 30, 32, 36, 37, 38,
It is solved by the features of 40 and 41. Further suitable embodiments are claimed in claims 2 to
25, 29, 31, 33, 34, 35, 39 and optionally 37 and 38 features.

According to the present invention there is provided a method of producing a channel-forming protein found in Gram-positive bacteria, wherein the channel-forming protein is: a) heterologous overexpressed, or b) purified from mycobacteria. A method is obtained, wherein the extraction temperature is above 50 ° C.

By channel-forming protein is meant a protein capable of forming a water-filled channel or a water-filled channel-like structure. Such proteins are naturally
It occurs especially in the cell wall of bacteria. They can form channel-like structures that are 3 nm in diameter or even larger. Channel-like structures or channels can be made up of a number of substructures, in particular 4 or 8 substructures.

This structure of the present invention is more efficient as compared to conventional approaches, offers the possibility of a wide range of automation of chromatographic purifications and allows a dramatic increase in yield. The Gram-positive bacterium can be a bacterium containing at least one mycolic acid.
In one embodiment, the bacterium is a mycobacteria, preferably Mycobacterium smegmatis.

The channel forming protein can be a porin. It is preferably chemically stable in organic solution and / or up to 80 ° C., more preferably 100
It is a porin that is thermostable at temperatures up to ° C. By stable is meant that the channel-like structure of the protein remains intact and essentially no denaturation of the protein occurs.

Preference is given to porin MspA, MspC, MspD, fragments of these porins, proteins homologous to these porins or proteins of sequences obtained from these porins. MspA corresponds to amino acids 28-211 of sequence 3 (see below), MspC corresponds to sequence 7 and MspD corresponds to sequence 9. The homologous protein of porin or a fragment thereof has a structure similar to that of the porin or a fragment thereof. At least 20% of the amino acids are identical or homologous to those porin or fragment amino acids. An amino acid in a protein is homologous to another amino acid if it can be replaced with another amino acid without affecting the function or structure of the protein. The protein deduced from the structure of porin may lose a single or a few amino acids compared to its sequence,
Alternatively it contains other amino acids or amino acid analogues.

The above-mentioned proteins are particularly suitable for the production of nanostructures because of their surprisingly high chemical and thermal stability. Good yields are obtained when the heterologous overexpression is carried out in E. coli or in mycobacteria. For overexpression, it is necessary to use a gene encoding a channel-forming protein, preferably a porin. For overexpression it is preferred to use the mspA gene according to sequence 1 (see below), the mspC gene according to sequence 6 or the mspD gene according to sequence 8. The mutant gene obtained from SEQ ID NO: 1, 6 or 8 was used for overexpression, whereby the mutation is essentially chemical and thermostable, and channel-like structures MspA, Msp
Such as essentially corresponds to that of C or MspD. The mutation is m
Such that the codon usage of the spA, mspC or mspD genes is adapted to the usage of highly expressed genes in E. coli. These codons are Nakamura,
T. et al. `` Two types of linkage between codon usage and gene-expression level
s. ”,“ FEBS Lett. ”289, 123-125 (1991).

The mutation in the sudden variant mspA, mspC or mspD gene is essentially
It can be used for overexpression if the G + C content is reduced to less than 66%. Codon usage adaptation dramatically improves overexpression of MspA, MspC and MspD in E. coli. The yield of the channel-forming protein MspA can be further increased by factors 10-20 via overexpression in E. coli as compared to the native protein production method described above.

It is useful to use the synmspA gene with sequence 4 for overexpression. An overexpression vector for E. coli in which the synmspA gene according to sequence 4 is inserted can be used for this purpose. Suitable vectors include Hanning, G. and Makri.
des, SC, "Trends in Biotechnology" 1998, Vol. 16, page 54, the contents of which are incorporated herein by reference.

It has also been beneficially found to harvest channel-forming proteins from the cell wall of Gram-positive bacteria by using nonionic or zwitterionic detergents. The detergent may be selected from the following group: isotridecyl poly (ethylene glycol ether) _n , alkyl glucosides, especially octyl glucoside, alkyl maltosides, especially dodecyl maltoside, alkyl thioglucosides, especially octyl thioglucoside, octyl-polyethylene oxide. And lauryl dimethylaminoxide. Phosphate buffer (100 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , pH 6.5, 150 mM NaCl
A critical micelle concentration (CMC) of more than 2 times in) is preferably used. Zwitterionic and nonionic detergents can be found in M. smegmatis (M. smegmat
dissolves the channel-forming protein MspA from the cell wall of is) very efficiently, yielding good yields.

It is further indicated that it is useful that the extraction temperature is 80-110 ° C., preferably 90-100 ° C., and / or the extraction time is 5-120 minutes, preferably 25-35 minutes. There is. Particularly preferred is the use of buffers having an ionic strength higher than 50 mM NaCl or Na-phosphate. Especially the extraction at 100 ° C. and the use of buffers with high ionic strength or the use of zwitterionic and nonionic detergents improve the extraction method of porin from Mycobacterium smegmatis. Compared with conventional techniques for purification of these proteins using organic solvents or their extraction at room temperature, it offers the following advantages: aa) does not require the use of organic solvents bb) other Minimal protein contamination cc) Efficient extraction.

It is also possible to purify MspA by dissolving it in dimethylsulfoxide at a temperature in the range 50-110 ° C., then cooling the solution to room temperature and filtering the MspA precipitate. Preferably, the channel-forming protein is precipitated for purification, especially with acetone. This approach results in further enrichment of MspA with respect to other non-precipitated proteins. It may also be beneficial to purify the protein using ion exchange chromatography methods, particularly anion exchange chromatography methods. Further purification can be accomplished using size exclusion chromatography.

Regeneration of channel-forming proteins produced by heterologous overexpression can be accomplished by increasing the local concentration of the protein. The increase can be achieved by electrophoretic enrichment, in particular by DC current, by sedimentation or by adsorption on a suitable surface (in particular a membrane). Useful is DC current at 50V for 30 minutes. Another aspect of the invention is a channel forming protein from Gram positive bacteria produced by the method of the invention.

Gram-positive bacteria are bacteria that contain mycolic acid, whereby it is beneficial to use mycobacteria, preferably Mycobacterium smegmatis. It is particularly beneficial that the channel forming protein is a porin that is chemically stable to organic solvents. Porins are essentially stable at temperatures up to 80 ° C, preferably up to 100 ° C. This thermal stability is dependent on MspA, MspC, MspD,
It is indicated by a fragment of these porins, a porin homologous to one of these fragments, or a sequence of porin (MspA, MspC, MspD) or a porin obtained from these fragments. The chemical and thermostability, and the channel-forming structure of the proteins thus obtained are, in general, MspA, MspC.
, Corresponds to the stability of the MspD protein. It is further possible that further proteins not mentioned here possess very similar properties and are therefore included within the scope of the present invention.

The channel forming protein of the present invention has the following advantages: aaa) The channel forming protein is Mycobacterium smegmatis (
If found in the cell wall of M. smegmatis), they can be dissolved in an organic solvent (eg CHCl ₃ / MeOH) without denaturation. The channel-forming property is
It remains in the organic solvent.

Bbb) They can be precipitated with acetone without denaturation. ccc) They survive without denaturation even during boiling in detergent (eg 10 min in 3% SDS). This extreme stability of the proteins of the invention against chemical and thermal denaturation allows them to be used to generate technically applicable nanostructures.

According to the invention, the gene encoding the channel-forming protein of the invention is further claimed. This is the mspA gene according to sequence 1 and ms according to sequence 6.
It can be the pC gene or the mspD gene according to sequence 8. As an additional substance, mutated mspA gene, mspC gene or msp
A D gene is provided, in which case the codon usage of said gene is practically the same as the codon usage of the highly expressed gene in E. coli. The mutation has a G + C content of 66.
% May be reduced to less than%. The mutated gene is further characterized by the chemical and thermostability of the expressed protein, and the channel-like structure of MspA, Ms
Obtained from one of sequences 1, 6 or 8 as being essentially similar to that of pC or MspD. Further mutations not mentioned herein are:
A person skilled in the art can think. Genes that result in the formation of the channel protein of the present invention, such as the mutated mspA gene, are within the scope of the claims appended hereto, in which case the mutated gene is synm according to sequence 4.
spA gene (see below).

Yet another object of the invention is the plasmid vector pMN501, and an overexpression system in which E. coli contains said plasmid vector. The following embodiments of the present invention will be described with reference to the drawings. In all gel electrophoresis experiments, in a sample buffer containing 40 mM Tris (hydroxymethyl) aminomethane, pH 7.0, 3% sodium dodecyl sulfate, 8% glycerol, 0.1% Selva Blue G for 30 minutes at room temperature, The proteins were incubated and then separated by gel electrophoresis by their size.

1a-c show that M. smegmati (M. smegmati) at different temperatures.
s) shows the proteins extracted from. Figure 1a shows a 10% denaturing polyacrylamide gel stained with Coomassie blue. Lane M: molecular weight markers (200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21
.5 and 14.4 kDa). Lanes 1-8: 30, 40, 50, 60, 70, 80, 90 and 100 ° C
12 μl of each extract obtained in.

FIG. 1b shows an immunoblot analysis of an 8% denaturing polyacrylamide gel blotted onto PVDF membrane. Proteins were visualized using MspA antiserum and chemiluminescence reaction (ECL detection system, AmershamPharmasia, Vienna, Austria). Lane M: molecular weight markers (97.4, 68, 46, 31, 20.1, 14.4 kDa). Lane 1 to 3:30
, 2 μl extract obtained at 40 or 50 ° C .; lanes 4-8: 1 μl extract obtained at 60, 70, 80, 90 or 100 ° C .; (9) 1 ng MspA.

FIG. 1 c shows a denaturing 8% polyacrylamide gel stained with silver. Lane M:
Molecular weight markers (200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, 14.4, 6 k
Da). Lanes 1 and 2: 15 μl extract obtained at 30 and 40 ° C. respectively; Lane 3: 15 μl extract obtained at 50 ° C .; Lanes 4-8: 60, 70, 80, 90 or 10
4 μl extract obtained at 0 ° C .; (9) 270 ng purified MspA.

[0030]   Figure 2 shows a denaturing 10% polyacrylamide gel stained with Coomassie blue.
.   Lane M: molecular weight markers (200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5
, 14.4, 6 kDa); Lane 1: POP05 buffer (100 mM Na).₂HPO_Four/ NaH ₂ PO_FourPH 6.5, 0.1 mM EDTA, 150 mM NaCl, 0.5% octylpolyethylene
Lenoxide (OPOE) obtained using Mycobacterium smegmatis
40 μg protein of extract from (M. smegmatis); lane 2: precipitated with acetone
40 μg protein of the extract after raining; Lane 3: anion exchange chromatography
And 4 μg of protein after pooling of fractions containing MspA; lane 4: a
4 μg protein of pooled MspA fraction after sedimentation with cetone; lane 5: size
4 μg tag after exclusion chromatography and pool of fractions containing MspA
Quality. mspA gene, mspA gene + promoter, and putative signal
The sequence of the MspA protein having the sequence No. is the sequence 1-3 in the sequence protocol.
Will be shown.

FIG. 3 shows the purification of the channel forming protein MspA from E. coli. Ten
Proteins were separated by their size in a% denaturing polyacrylamide gel. The gel was stained with Coomassie blue. Lane 1: Lysate from E. coli BL21 (DE3) / pMN501 before induction with IPTG. Lane 2: Lysate from E. coli BL21 (DE3) / pMN501 after induction with IPTG. Lane 3: molecular weight markers (200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, 14.4, 6 kDa
). Samples were incubated at 37 ° C for 30 minutes before loading on the gel.

FIG. 4 schematically shows the construction of vector pMN501 for overexpression of E. coli BL21 (DE3). The abbreviations have the following meanings: lacI: gene encoding a lactose repressor. nptI: gene encoding neomycin phosphotransferase. This gene confers resistance to kanamycin. Ori: Origin of replication. RBS: Ribosome binding site.

FIG. 5 schematically shows an apparatus for the regeneration of the monomer MspA. A 5 cm long polyethylene disposable pipette tip was shortened by 2 mm at its lower end. The tip was filled with 1.7% agarose in TAE buffer. Pencil (brand: Eberhard
Faber, 3H) core lead shortened to 5 cm. 5 μg in polypropylene tube without lid
60 .mu.l of a solution containing modified MspA of 3. A pipette tip and core lead were placed in the solution and connected as the cathode and anode, respectively.

FIG. 6 shows regeneration of denatured MspA. Schagger (Schagger, H. and von Jagow,
G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis fo
Proteins were separated in 10% denaturing polyacrylamide gels as described by Anal Biochem 166, 368-79 (1987)). The gel was stained with silver. Lane M
: Molecular weight markers (116, 97, 66, 55, 36.5, 31, 21.5, 14.4 kDa). Lane 1
: 800 ng modified MspA. Lane 2: 800 ng MspA after regeneration reaction. Samples were incubated at 37 ° C for 30 minutes before loading on the gel.

Figures 7a-7c show electron micrographs of modifications of the channel forming protein MspA from Mycobacterium smegmatis. Sample preparation was carried out as follows: 1 ml solution of MspA (c (MspA) = 17.2 × 10 ⁻⁹ mol / L, 100 mM Na ₂ HP.
O ₄ / NaH ₂ PO ₄ , pH 6.5, 150 mM NaCl, 0.10 g / L SDS) at 24.5 ° C.
And ultrasonically dispersed in a water bath. The distance between the liquid and the HOPG (carbon) surface (1.0 mm ² ) was 5.0 cm. The dispersed liquid droplets were contacted with the HOPG surface for 20 minutes.

FIG. 7a shows an isolated channel forming protein. FIG. 7b demonstrates a ribbon-like structure showing large holes with a diameter of 12 nm. Figure 7c shows two types of channels in a ribbon-like structure: a channel with a small diameter of about 2.4 nm and about 9-10 nm.
Is a channel having a large diameter.

[0037]   Example 1: M. smegmatis at different temperatures
Extraction of MspA from   10 milligrams of mc of M. smegmatis²
155 cells (wet weight) in PBS (100 mM sodium phosphate, pH 7.0, 150 mM N
aCl, 0.1 mM EDTA) and 150 μl PG05 buffer (0.5% isotope).
Lydecyl polyethylene glycol ether, 100 mM Na₂HPO_Four/ NaH₂PO _Four , 0.1 mM EDTA, 150 mM NaCl, pH 6.5). Fine resuspension
Vesicles were incubated at 30, 40, 50, 60, 70, 80, 90 or 100 ° C for 30 minutes.
Samples were cooled on ice for 10 minutes and centrifuged at 4 ° C for 10 minutes. Evaporate the volume of the supernatant
The product was reduced from 120 μl to 10 μl. Gel electrophoresis, as shown in FIGS.
Proteins were separated by their size by motion. Total protein in the extract
The fraction of MspA compared to quality is significantly increased at temperatures above 50 ° C. Those
At this temperature, other proteins are only extracted in small amounts.

Example 2: Msp from Mycobacterium smegmatis
Purification of A 10 grams of M. smegmatis cells in PBS
Washed with water, resuspended in 35 mL of POP05 and boiled in a water bath for 30 minutes with stirring. The cell suspension was cooled on ice for 10 minutes and centrifuged at 27,000 g for 15 minutes at 4 ° C. 42 mL of supernatant was gently mixed with an equal volume of ice cold acetone. 1 this mixture on ice
Hold for time and centrifuge at 8000g for 15 minutes at 4 ° C. 10 mL of precipitated protein 25 mM
N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEP
ES), pH 7.5, dissolved in 10 mM NaCl, 0.5% OPOE (AOP05),
Anion exchange column "POROS 20HQ" (Persepti) with a volume of 1.7 mL
ve Biosystems, Cambridge, USA). After washing the column with 14 mL of AOP05, the bound protein was washed with 100% AOP05 to 100% BOP05 (25 mM HEPES).
, PH 7.5, 2 M NaCl, 0.5% OPOE), eluting over 34 mL. 1 mL of 90 fractions were collected and analyzed by gel electrophoresis. The four fractions with the highest amount of MspA were pooled and precipitated with acetone as before. 6 pellets
Insoluble material was removed by dissolving in 00 μl of AOP05, incubating on ice and centrifuging at 4 ° C. for 5 minutes. The protein solution was loaded onto a gel filtration column "Superdex G200" (Pharmacia, Freiburg, Germany) with a volume of 24 mL. The protein was eluted with 48 mL of AOP05 at a rate of 0.2 mL / min. 1 m
Fifty fractions of L were collected and analyzed using a denaturing polyacrylamide gel stained with silver. Fractions containing apparently pure MspA were pooled. The purification steps are shown in FIG. The yield was 700 μg. 1 μg of this sample probe did not show any contamination of other proteins in the silver stained denaturing polyacrylamide gel (data not shown). Therefore, MspA was apparently purified to homogeneity.

Example 3: Purification strategy for the channel-forming protein MspA from E. coli To further increase the yield of MspA, overexpression of the mspA gene in E. coli is suggested. The mspA gene is a Mycobacterium smegmatis (
M. smegmatis). The T7 expression system is selected for overexpression of the mspA gene. By PCR, plasmid pPO
The mspA gene was amplified from R6. All codons in the native mspA sequence, which rarely occur in highly expressed genes, were replaced. All mutations are listed in Sequence 4 of the sequence protocol (see below). Stemmer (Stemmer, WP, Cameri, A., Ha,
KD, Brennan, TM and Heyneker, HL Single-step assembly of a gene a
nd entire plasmid from large numbers of oligodeoxyribonucleotides. Gene
164, 49-53 (1995)), which synthesized this gene by oligonucleotide assembly and named synmspA. The synmspA gene replaces the mspA gene in vector pMN500, but its use did not provide detectable amounts of MspA in E. coli in vector pMN501 (Figure 4). The vector pMN501 was transformed into E. coli BL2 after induction with IPTG.
It caused strong expression of MspA monomer (20 kDa) in 1 (DE3). This protein is called recombinant MspA (rMspA) and carries sequence 5 of the sequence protocol (see below).

Example 4: Purification procedure of channel-forming protein MspA from E. coli mit E. coli B in 1 liter LB medium containing 30 μg / mL kanamycin
L21 (DE3) / pMM501 was inoculated and the culture was grown at 37 ° C. to an OD ₆₀₀ of 0.6. The cells were then induced with 1 mM IPTG and incubated at 37 ° C. for an additional 6 hours until the culture reached an OD ₆₀₀ of 2.2. Harvest cells by centrifugation and 40 mL
Resuspended in A-buffer (25 mM Hepes, pH 7.5, 10 mM NaCl)
It was dissolved by boiling in water for 10 minutes. Keep the cell lysate on ice for 10 minutes,
Cell debris and insoluble proteins were precipitated by centrifugation at 10,000 g for 10 minutes. Anion exchange chromatography (POROS HQ20, Perseptive Biosys
tems, Cambridge, USA), and a linear gradient of 10 mM to 2 M sodium chloride was used to fractionate the supernatant. Monomeric MspA elutes at 350 mM NaCl. Msp
Fractions containing A were pooled. MspA is purified from proteins with large molecular weight using size exclusion chromatography (Superdex G200, Pharmacia, Freiburg, Germany). Yield is 10 mg MspA, purity> 95% (data not shown).

Example 5: Electrochemical assembly of the channel forming protein MspA Overexpression of MspA in E. coli facilitates production of MspA in good yield. However, the large fraction of purified protein is inactive. Regeneration of MspA to its active form can be accomplished using the following protocol: Regeneration can occur in a device specifically designed for this purpose (Figure 5). The regeneration reaction is carried out with 5 μg of the monomer MspA in the above device by applying a voltage of 50 V for 30 minutes. The sign of the applied voltage is then reversed for 5 seconds to remove the porins adsorbed on the lead surface. After the refolding reaction, porin is analyzed on a denaturing polyacrylamide gel (Fig. 6). This gel shows that a large fraction of proteins assemble into oligomers. This assembled MspA is demonstrated to have high channel-forming activity by reconstitution in lipid bilayer experiments. This experiment demonstrates that regeneration of the monomer MspA is possible with a small DC voltage. This regeneration reaction is very easy to carry out and functional Ms due to overexpression of E. coli.
It is an important component of pA purification.

[0042]   Sequence list:   1. mspA gene (translated)   2. mspA gene + promoter (translated)   3. MspA protein with putative signal sequence   4. synmspA gene (translated)   5. rMspA protein   6. mspC gene   7. MspC protein   8. mspD gene   9. MspD protein

[Sequence list]

[Brief description of drawings]

FIG. 1a: Mycobacterium smegmatis (M. sme) as shown by gel electrophoresis.
temperature dependent extraction of MspA from gmatis).

FIG. 1b: Mycobacterium smegmatis (M. sme) as shown by gel electrophoresis.
temperature dependent extraction of MspA from gmatis).

FIG. 1c: Mycobacterium smegmatis (M. sme) as shown by gel electrophoresis.
temperature dependent extraction of MspA from gmatis).

Figure 2: Mycobacterium smegmatis (M. sme) as shown by gel electrophoresis
purification of MspA from gmatis).

[Figure 3]   Purification of MspA from E. coli as shown by gel electrophoresis.

[Figure 4]   Construction of plasmid vector pMN501.

[Figure 5]   1 is a schematic diagram showing an apparatus for regenerating monomer MspA.

[Figure 6]   Regenerated MspA as shown by gel electrophoresis.

FIG. 7a   Modification of the channel forming protein MspA as shown by electron microscopy.

FIG. 7b   Modification of the channel forming protein MspA as shown by electron microscopy.

FIG. 7c   Modification of the channel forming protein MspA as shown by electron microscopy.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F-term (reference) 4B024 AA03 CA02 EA04 GA25 HA01                 4B064 AG01 CA02 CC24 CE07 CE11                       CE15 DA16                 4H045 AA10 AA20 CA11 EA34 EA45                       FA74 GA05 GA10 GA22 GA23                       GA30

Claims (41)

[Claims] 1. A method for producing a channel-forming protein found in Gram-positive bacteria, wherein the channel-forming protein is obtained by: a) heterologous overexpression, or b) purification from mycobacteria and at an extraction temperature. Is higher than 50 ℃. 2. The method according to claim 1, wherein the Gram positive bacterium contains at least one mycolic acid. 3. The method according to claim 1, wherein the bacterium is mycobacteria, preferably Mycobacterium smegmatis. 4. The channel-forming protein is porin.
The method described in any one of. 5. The method of any of claims 1-4, wherein the porin is essentially chemically stable to organic solvents. 6. The method according to claim 1, wherein the porin is essentially thermostable at temperatures up to 80 ° C., preferably up to 100 ° C. 7. Porin MspA, MspC, MspD, a fragment of one of these porins, a homologous protein from one of these porins or a protein obtained from the sequence of one of these porins. The method according to any one of claims 1 to 6, wherein 8. The method according to claim 1, wherein the heterologous overexpression is realized in E. coli or mycobacteria. 9. The method according to claim 1, wherein a gene encoding a channel forming protein, preferably porin, is used for overexpression. 10. The mspA gene according to sequence 1 and the mspC according to sequence 6.
The method according to any of claims 1 to 9, wherein the gene or the mspD gene described in Sequence 8 is used for overexpression. 11. A mutant gene obtained from SEQ ID NO: 1, 6 or 8 is used for overexpression, the mutation being essentially chemical and thermostable, and channel-like structures MspA, MspC. 11. A method according to any of claims 1 to 10 which is essentially the same as that of MspD. 12. The mutation is essentially mspA, mspC or mspD.
The method according to any one of claims 1 to 11, wherein the codon usage of the gene is adapted to the usage of a highly expressed gene in Escherichia coli. 13. The method according to claim 1, wherein the mspA, mspC or mspD gene is used for overexpression and the mutation is essentially such that the G + C content is reduced to less than 66%. the method of. 14. The method according to any one of claims 1 to 13, wherein the synmspA gene set forth in Sequence 4 is used for overexpression. 15. The method according to any of claims 1 to 14, wherein a suitable vector for overexpression in E. coli containing the synmspA gene according to SEQ ID NO: 4 is used. 16. The method according to claim 1, wherein the channel-forming protein is produced from cell walls from Gram-positive bacteria using a nonionic or zwitterionic detergent. 17. The detergents used are listed below: isotridecyl poly (ethylene glycol ether) _n , alkyl glucosides, especially octyl glucoside, alkyl maltosides, especially dodecyl maltoside, alkyl thioglucosides, especially octyl thioglucoside, 17. A method according to any of claims 1 to 16 obtained from octyl-polyethylene oxide and lauryl dimethylaminoxide. 18. The method according to claim 1, wherein the extraction temperature is 80 to 110 ° C., preferably 90 to 100 ° C. 19. Extraction time of 5 to 120 minutes, in particular 25 to 35 minutes.
The method described in any one of. 20. The method according to claim 1, wherein a buffer having an ionic strength higher than 50 mM NaCl or Na-phosphate is used. 21. The method according to claim 1, wherein the channel-forming protein is purified by precipitation, in particular with acetone. 22. The method according to claim 1, wherein the channel-forming protein is purified by ion exchange chromatography, in particular anion exchange chromatography.
The method according to any one of 21. 23. The method of any of claims 1-22, wherein the channel forming protein is purified using size exclusion chromatography. 24. The method according to claim 1, wherein the channel-forming protein produced by heterologous overexpression is regenerated by increasing the local concentration. 25. The method according to claim 24, wherein the increase in local concentration is achieved by electrophoretic enrichment, in particular by DC current, by sedimentation or by adsorption on a suitable surface, in particular on a membrane. 26. A channel-forming protein from a Gram-positive bacterium produced by the method according to any one of claims 1 to 25. 27. A channel-forming protein from a Gram-positive bacterium wherein the channel-forming protein is a porin that is essentially chemically stable to organic solvents. 28. A channel-forming protein from a Gram-positive bacterium wherein the channel-forming protein is a porin which is essentially stable at temperatures up to 80 ° C. 29. The channel-forming protein of claim 28, wherein the channel-forming protein is a porin that is essentially stable at temperatures up to 100 ° C. 30. The channel-forming proteins are porin MspA and Msp.
C, MspD, a fragment of one of these porins, a protein homologous to these porins or fragments thereof, or a protein obtained from these porins, which is a channel-forming protein from Gram-positive bacteria. 31. The channel-forming protein of claim 30, wherein the channel-like structure of the chemical and thermostability and putative proteins is essentially that of the protein MspA, MspC or MspD. 32. A gene encoding the channel-forming protein according to any one of claims 26 to 31. 33. The gene according to claim 1, which is the mspA gene according to Sequence 1.
The described gene. 34. The gene according to claim 6, which is the mspC gene according to Sequence 6.
The described gene. 35. The method according to claim 32, wherein the gene is the mspD gene according to Sequence 8.
The described gene. 36. The mutation is essentially mspA, mspC or mspD.
A mutated mspA, mspC or mspD gene whose codon usage of the gene is such that it is adapted to that of a highly expressed gene in E. coli. 37. In particular, the mutated mspA gene according to claim 36, ms
pC or mspD genes with essentially less than 66% G mutations
A gene consisting of + C content reduction. 38. A mutated mspA, mspC or mspD gene according to claim 36 or 37, obtained from one of sequences 1, 6 or 8, wherein the mutations are chemical and thermal of the protein. Genes whose stability, as well as channel-like structures, are of MspA, MspC or MspD for all practical purposes. 39. The mutated mspA gene according to claims 36 to 38, wherein the mutated gene is the synmspA gene according to Sequence 4. 40. A plasmid vector pMN501. 41. An overexpression system in which E. coli contains the plasmid vector pMN501.