KR101018878B1 - Refolding and reconstitution of human recombinant Bax inhibitor-1 into liposomes from inclusion bodies expressed in Escherichia coli - Google Patents

Abstract

The present invention relates to the simultaneous refolding and reconstitution of recombinant Bax inhibitor-1 (BI-1) from inclusion bodies expressed in E. coli. Functional analysis showed that the resulting proteoliposome responds to an acidic environment and induces the release of inclusion Ca ²⁺ from liposomes. The secondary structure of the reconstituted BI-1, determined by circular dichroism spectroscopy, demonstrated an increase in α-helix content and a decrease in random structure upon exposure to an acidic environment. This structural change may be responsible for proton-induced Ca ²⁺ release of BI-1.

Bax inhibitor-1, liposomes, refolding, reconstitution.

Description

Refolding and reconstitution of human recombinant Bax inhibitor-1 into liposomes from inclusion bodies expressed in Escherichia coli}

The present invention relates to refolding of human recombinant Bax inhibitor-1 and to liposomes comprising said refolded human recombinant Bax inhibitor-1.

The present invention is derived from research conducted as part of the Basic Research Projects Support Project of the Korea Research Foundation. [Task No .: KRF-2008-314-C00231, Title: Biological of microsomal monooxygenase (MMO) in apoptosis process Role and cross-talk between mitochondria and endoplasmic reticulum]

Bax inhibitor-1 (BI-1; also known as "testis enhanced gene transcript") is an anti-apoptotic protein that can inhibit Bax activation and translocation to mitochondria (1). An ubiquitously expressed protein consists of 237 amino acids and has a molecular weight of about 26 kDa. BI-1 also contains several transmembrane domains and is present in the endoplasmic reticulum (ER). Sequence homology between species shows that characteristic hydrophobicity and ER membrane localization are evolutionarily conserved (2). A biological function, BI-1 is Ca ²⁺ - sensitive, ER- target fluorescent proteins and Ca ²⁺ - affects the leakage of calcium, from the ER as measured by sensitive dye (3). Recently, we have proposed that BI-1 can act as a pH-dependent regulator of Ca ²⁺ channels in ER (4). However, the exact biological role of BI-1 is still unknown and thus recombinant BI-1 is required to clarify protein function in vitro.

One embodiment provides a method for efficiently producing liposomes comprising the BI-1 protein.

Another embodiment provides liposomes comprising the BI-1 protein produced by the above method.

One embodiment includes separating an inclusion body comprising a BI-1 protein from a prokaryotic cell culture;

Dissolving the isolated inclusion body in a first buffer comprising a first concentration of chaotropic agent, a reducing agent and an amphoteric surfactant;

Mixing the first buffer solution with phospholipids;

Exchanging a first buffer in the mixture for a second buffer, the second buffer comprising a chaotropic agent at a second concentration lower than the first concentration, and the reducing agent and no amphoteric surfactant Not;

Exchanging the second buffer solution with a third buffer, wherein the third buffer comprises the reducing agent and does not include the chaotropic agent and the amphoteric surfactant; And

It provides a method for producing a liposome comprising a BI-1 protein, comprising; separating the liposome comprising a BI-1 protein from the third buffer solution.

The method includes separating an inclusion body comprising the BI-1 protein from prokaryotic cell culture. The prokaryotic cell may be Escherichia coli. The culture may be cultured prokaryotic cells transformed with a vector comprising a nucleic acid encoding a BI-1 protein under conditions such that the nucleic acid is overexpressed. For example, the vector may be controlled by a lac operon, and the condition may be a condition including an expression inducer, for example, IPTG. Separation of the inclusion body containing the BI-1 protein may be accomplished by methods known in the art. For example, the inclusion bodies can be isolated by disrupting the cells, centrifuging the cell debris and separating the precipitated pellets. The crushing may be to crush the culture by suspending and pressing in a buffer. The compacted crushed material can be centrifuged to recover the pellet, and the recovered pellet can be added to the buffer and suspended by sonication. The BI-1 protein may be a human BI-1 protein having an amino acid sequence as set forth in Genbank accession number P55061-1 (SEQ ID NO: 1). The nucleic acid encoding the human BI-1 protein encodes the amino acid sequence of Genbank accession number P55061-1, and may have, for example, a nucleotide sequence of SEQ ID NO: 2.

The method also includes dissolving the isolated inclusion body in a first buffer comprising a first concentration of chaotropic agent, a reducing agent, and an amphoteric surfactant.

The dissolution may be accomplished by incubation with or without shaking at a suitable temperature. For example, the temperature may be 2 ℃ to 40 ℃. Incubation time can be appropriately determined by those skilled in the art. For example, the incubation time may be 1 hour or more.

The chaotropic agent may be urea or guanidinium chloride. The chaotropic agent of the first concentration may be 6M to 8M urea or 6M or more guanidinium chloride.

The reducing agent may be one selected from the group consisting of DTT (dithiothritol), cysteine, 2-mercaptoethanol, and TCEP (tris (2-carboxyethyl) phosphine). The concentration of the reducing agent is a concentration capable of maintaining the thiol group in a reduced state, and can be appropriately determined according to the reducing agent selected by those skilled in the art. For example, in the case of DTT, it may be 0.5mM to 2mM. The amphoteric surfactants include CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate), CHAPSO (3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate), cholate, and It may be selected from the group consisting of oxycholate. The concentration of the amphoteric surfactant can be appropriately determined according to the amphoteric surfactant selected by those skilled in the art. For example, the concentration of the amphoteric surfactant may be 0.5% (w / v) to 3.0% (w / v).

The first buffer may be a buffer containing 8M urea, 0.1 mM DTT and 1.5% (w / v) CHAPS. For example, the first buffer may be a buffer consisting of 8M urea in water, 0.1 mM DTT and 1.5% (w / v) CHAPS. More specific examples of the first buffer may be a buffer comprising 20 mM HEPES buffer (pH 7.4), 8M urea, 0.1 mM DTT, 1 mM CaCl ₂ and 1.5% (w / v) CHAPS in water.

The first buffer including the chaotropic agent, the reducing agent, and the amphoteric surfactant may be HEPES buffer, Tris-HCl buffer or phosphate buffer.

The first buffer may further include CaCl ₂ . By reconstituting the liposomes in a buffer containing CaCl ₂ , Ca ²⁺ can be contained within the final liposome.

The method also includes mixing the first buffer solution with phospholipids. The phospholipid is hydrated by the mixing. The phospholipid is not limited as long as it can constitute a biofilm. For example, the phospholipid may be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The ratio of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine may be in a molar ratio of 5: 3: 2. The phospholipid may be one of dry air. The concentration of the phospholipid may be greater than 0M and less than or equal to 5 mM.

The method also includes exchanging a first buffer in the mixture with a second buffer, the second buffer comprising a second concentration of the chaotropic agent and a reducing agent that is less than the first concentration and wherein the amphoteric The surfactant includes a step that is not included.

The exchange may be performed by dialysis or ultradialysis. The chaotropic agent of the second concentration may be 1.5M to 5M urea or 1M to 4M guanidinium glolide. The second buffer may be the same as the first buffer except for the low concentration of the chaotropic agent and no amphoteric surfactant. The second buffer may be a buffer including 20mM HEPES buffer (pH 7.4), 2M urea, 0.1mM DTT and 1mM CaCl ₂ .

The method also includes exchanging the second buffer solution with a third buffer, wherein the third buffer includes the reducing agent and does not include the chaotropic agent and the amphoteric surfactant. The exchange may be performed by dialysis or ultradialysis. The third buffer may be the same as the second buffer except for including the reducing agent and the chaotropic agent and the amphoteric surfactant. The third buffer may be a buffer including 20mM HEPES buffer (pH 7.4), 0.1mM DTT and 1mM CaCl ₂ .

The method may also further comprise washing the separated liposomes. The washing can be done by soaking the separated liposomes in an appropriate washing buffer. Examples of such wash buffers include HEPES buffers, Tris-HCl buffers and phosphate buffers. The wash buffer may be a buffer consisting of 20mM HEPES buffer (pH 7.4), 100mM NaCl, 0.1mM DTT, 0.5mM EGTA and 1M KCl.

The method further includes, after washing the separated liposomes, exchanging the wash buffer in the liposomes using a buffer having the same components except that some components of the wash buffer have been removed. can do. The exchange can be dialysis or ultradialysis. The buffer used for the exchange may be a buffer from which 0.5 mM EGTA and 1M KCl have been removed from the wash buffer.

The method may further comprise performing cation exchange chromatography on the separated liposomes. The cation exchange chromatography may be appropriately selected depending on the cation selected. For example, Chelex 100 (Bio-Rad, Hercules, CA) can be used to remove free Ca ²⁺ .

Another embodiment provides a composition comprising a liposome comprising a BI-1 protein prepared by the above method. The above method is as described above. The BI-1 protein may be one having an amino acid sequence having the Genbank accession no P55061-1 (SEQ ID NO: 1).

The present invention provides a method of refolding recombinant BI-1 from an inclusion body and reconstituting it into liposomes. The proteoliposomes obtained by the process of the invention react to acidity and induce Ca ²⁺ efflux. Based on structural studies, acid-mediated Ca ²⁺ release is accompanied by structural changes in the BI-1 protein. The method of the present invention can be used for in vitro functional analysis of BI-1.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Materials and methods

material

All phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). Fluorescent Ca ²⁺ indicator, indo-1, and pET12a vectors were purchased from Invitrogen (Carlsbad, CA). Restriction enzymes were purchased from Takara-Bio (Shiga, Japan).

Way

Construction of Bax Inhibitor-1 Expression Plasmids

PET12a / BI-1 was generated by subcloning human BI-1 cDNA into a pET12a expression vector using polymerase chain reaction.

PCR reactions were designed such that the forward and reverse primer oligonucleotides included BamH1 and HindIII restriction enzyme recognition sites, respectively (forward: 5'-GACGGATCCATGAACATATTTGATCGAAAG-3 '(SEQ ID NO: 3), reverse: 5'-GACAAGCTTTTATTTCTTCTCTTTCTTCTT-3' (SEQ ID NO: 3) Number 4)). PCR was performed with pfu polymerase (Stratagene, LaJolla, CA). Prior to the addition of the polymerase, the reaction mixture was preincubated at 94 ° C. for 5 minutes, followed by an amplification cycle of 30 seconds at 90 ° C., 90 seconds at 60 ° C., and 3 minutes at 72 ° C. Was performed twice. The resulting PCR product was purified and treated with BamH1 and HindIII and ligated to pET-12a vector treated with the same restriction enzyme. Nucleotide sequences of the entire region containing the BI-1 gene were analyzed by dideoxy sequencing.

Expression of Recombinant BI-1 Protein

BL21 (DE3) containing pET12a / BI-1 was incubated in 500 ml of LB / ampicillin (50 μg / ml) at 37 ° C. until an absorbance of 0.5 (600 nm) was reached. Thereafter, isopropyl-β-D-thiogalactopyranoside (0.5 mM) was added and incubated for 4 hours to induce the expression of the recombinant protein. To obtain the inclusion of expressed BI-1, the obtained bacterial pellet (ca. 4 g of cell wet weight) was added to 20 ml of lysis buffer (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM phenylmethyl). Sulfonyl fluoride, and 5 μg / ml benzamide, leupetin, and pepstatin). The cells were lysed through Amicon French pressure cell (Millipore, Billerica, Mass.) And the lysates were centrifuged (12,000 × g, 10 min, 4 ° C.) to recover the pellets. The pellet was resuspended in 5 ml of lysis buffer using probe sonicaiton and centrifuged (12,000 × g, 10 min, 4 ° C.). The same step (resuspension by sonication and centrifugation) was repeated five times. The inclusion bodies-containing pellets finally obtained were placed in Buffer A consisting of 20 mM HEPES (pH 7.4), 8M urea, 0.1 mM dithiothreitol (DTT), 1 mM CaCl ₂ , and 1.5% (w / v) CHAPS. The solution was dissolved by incubating at 25 ° C with gentle shaking.

Refolding and Reconstitution of BI-1 from Protoplasts

The chloroform solution of lipids was stored in sealed ampoules at -20 ° C under argon gas. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (all derived from bovine brain) dissolved in chloroform were each mixed in a molar ratio of 5: 3: 2. Phospholipid concentrations were determined by phosphorus assay (5). Phospholipid concentrations were increased to 5 mM to reconstitute the BI-1 protein. The solvent was evaporated under the flow of argon gas and residual chloroform was removed by centrifugal lyophilization by centrifugation. The dried lipids were hydrated with 1 ml of Buffer A containing about 200 μg of BI-1. The mixture was dialyzed for 12 h against excess of Buffer B (Buffer A without CHAPS) containing 2M urea. The dialysis step was repeated for an excess of buffer C (buffer B without urea) for 12 hours. The resulting proteoliposomes were precipitated by centrifugation at 12,000 × g for 40 minutes at 4 ° C. and buffer D (20 mM HEPES, pH 7.4, 100 mM NaCl, 0.1 mM DTT, 0.5 mM EGTA, and 1 M KCl). ) And dialyzed for 12 hours at 4 ° C. against Buffer E (Buffer D excluding 1M KCl and 0.5 mM EGTA). The resulting proteoliposomes were passed through Chelex 100 (Bio-Rad, Hercules, CA) to remove free Ca ²⁺ . The formation of proteoliposomes was monitored by spectroscopy (6) by measuring the absorbance at 450 nm during dialysis as described above (6). The amount of reconstituted BI-1 protein was determined using the NanoOrange® Protein Quantitation Kit (Invitrogen).

Indo-1 Fluorescence and ⁴⁵ Ca ²⁺ Ion-mediated Ca from proteoliposomes, using ²⁺  Release

All experiments measuring Ca ²⁺ release from ^{proteoliposomes} were performed as described above (4). In summary, proteoliposomes are rapidly diluted with an acidic solution (50 mM sodium phosphate, pH 6.5, or 50 mM sodium citrate, pH 6.0 and 5.5) and a ratio of 1: 2 (v / v) followed by external delivery (indo Ca ²⁺ effluent was observed by measuring the fluorescence change of) -1 (5 μM). Fluorescence intensities were measured at emission and excitation wavelengths of 393 nm and 355 nm, respectively. To quantify proton-mediated Ca ²⁺ release from proteoliposomes, the acid-induced fluorescence intensity of Indo-1 was determined by fluorescence intensity after addition of Triton X-100 (final concentration 1% (v / v)). Compared. In addition, the proteosome liposomes were prepared in the presence of ⁴⁵ Ca ²⁺ to Ca ²⁺ in the nest ⁴⁵ under the same conditions described above ~ 20,000 cpm. After dilution of the proteoliposomes with acidic solution to induce the outflow of Ca ²⁺ , the samples were centrifuged (12,000 × g, 40 min, 30 ° C.). Scintillation counting was used to determine proton-mediated Ca ²⁺ release by radioactivity of the pellet and supernatant.

Circular Dichroism Spectroscopy

CD spectra were monitored in the far-ultraviolet range at 30 ° C. with a Jasco J-715 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan). The optical path length is 0.1 cm and the measurements are performed in acidic solutions containing either buffer E or proteothiosomes. Blanks (lipid with or without buffer) were recorded regularly and subtracted from the original spectra. On average, data from 20 scans were accumulated. Results were expressed as mean residue ellipticity based on the number of amino acids per BI-1 molecule after determination of protein concentration.

Antibody Agents Against BI-1 article

An epitope corresponding to the C-terminus of BI-1 containing additional Cys residues (CAMNEKDKKKEKK: SEQ ID NO: 5) was synthesized by PepTron (Daejon, Korea), and the keyhold limpet hemocyanin- Peptide complex method) was used as an antigen for producing antibodies in rabbits (7). Based on the prediction by the Lasergene ™ 6 program (DNASTAR, Madison, Wis.), This peptide was chosen as the only epitope region.

Example 1: Expression, Refolding, and Reconstitution of BI-1

Previously, we reported the preparation of human recombinant BI-1 and reconstitution into liposomes in a functional study using soluble fractions expressed in E. coli (4). However, only 25-50 μg / L culture could be obtained as soluble protein, and judging by the formation of precipitate, a significant amount of BI-1 was in an insoluble state. In this study, we report a method of simultaneously refolding recombinant BI-1 using inclusion bodies expressed in E. coli. The inclusions obtained were dissolved in a solution containing 8 M urea and 1.5% (w / v) CHAPS, neutral surfactant. Purity and identification of the lysed protein samples were performed by immunoblotting with antibodies to the C-terminal regions of SDS-PAGE and BI-1 (FIG. 1A). The inventors were able to obtain about 20-30 mg / L culture of inclusion bodies of recombinant BI-1, as estimated by determination of protein concentration and purity. Without additional purification steps, purity reached at least about 90% and refolding and reconstitution of BI-1 was performed as described. During dialysis for proteoliposome formulation, it is important to remove urea step by step for effective reconstitution (BI-1 dissolved in 8 M urea was dialyzed against a buffer containing 2 M urea). When the dialysis step was carried out in a buffer containing no 2 M urea, a significant amount of BI-1 was precipitated again. Instead of urea, we were able to obtain similar results when using 6M guanidine hydrochloride (GdnHCl) to dissolve the inclusion bodies (results not shown) (similar amounts of reconstituted BI-1 and Ca ²⁺ Similar effect for functional analysis as a channel: see FIG. 2). Stepwise dialysis is important for functional reconstitution of BI-1. In gradual dialysis, the lysed protein was dialyzed against a buffer containing 1.5 M GdnHCl to prevent remodeling of the inclusion bodies. In addition, CHAPS is required to reconstitute BI-1 into liposomes, and the amount of reconstituted BI-1 was significantly reduced using Buffer A, which does not contain surfactant. After preparation of the proteoliposomes, when analyzed as known (8), the average diameter of liposomes was determined to be about 360 nm (results not shown). During the dialysis and ultracentrifugation process to formulate proteoliposomes, if insoluble materials are present, they may precipitate with the proteoliposomes. However, when the resulting proteoliposomes were analyzed by the dynamic light scattering method as described above, the scattering values varied within the error range. Therefore, it could be concluded that proteoliposomes are the major products after dialysis and centrifugation steps.

To investigate the phospholipid concentration-dependent reconstitution of BI-1, the lipid concentration was increased to 5 mM in the presence of 200 μg / ml of BI-1 dissolved in Buffer A. Reconstituted BI-1 increased with increasing phospholipid concentration and reached a plateau near 2.5 mM (FIG. 1B). It was also calculated that about 64% of BI-1 dissolved in urea solution was reconstituted into liposomes.

Example 2: Hydrogen Ion-mediated Ca from Proteoliposomes ²⁺  Release

Functional assays were performed using proteoliposomes to monitor proton-mediated Ca ²⁺ efflux from liposomes to confirm the effectiveness of the methods of the present invention. Rapid dilution in the acidic solution of the BI-1 reconstituted vesicles leads to the release of the entrapped Ca ²⁺ (FIG. 2), as suggested previously (4), BI-1 activity as Ca ²⁺ channel Confirmed. The amount of Ca ²⁺ released was similar when analyzed by fluorescent dyes and ⁴⁵ Ca ²⁺ . These results suggest that increased acidity is associated with increased release of Ca ²⁺ by proteoliposomes. The results of the present invention also suggest that refolding and reconstitution methods using the inclusion bodies of BI-1 can be used for functional analysis of these proteins.

Example 3: Acid-Induced Structural Changes of Reconstituted BI-1

BI-1 has a transmembrane domain and can potentially function as a membrane protein (2, 9). To study the structural properties of membrane-embedded (BI-1) BI-1 embedded in the membrane and to correlate potential structural changes of BI-1 with acid-induced Ca ²⁺ efflux, we used a secondary CD to The structure was measured. 3 shows the CD spectrum of BI-1 reconstituted with proteoliposomes. When the proteoliposomes were suspended in a pH 7.4 solution, using the curve-fit method (10), the content of α-helix, β-sheet and random structures were 42%, 38% and 20%, respectively. It was estimated. Interestingly, the suspension of reconstituted BI-1 in acidic solution resulted in a significant increase in α-helix content and a decrease in random structure, with little change in β-sheet observed.

Table 1. Secondary structure estimation of BI-1 reconstituted with liposomes

pH α-helix% β-sheet% Random coil% 7.4 42 38 20 6.5 52 36 12 5.5 58 37 5

The data in Table 1 above was obtained by curve fitting to the previously disclosed (10) reference spectrum.

Formation of intramolecular disulfide bonds is an important structural feature of many proteins and significantly affects the folding process of recombinant proteins. BI-1 has five cysteine residues in the amino acid sequence and thus disulfide bonds may be present, although the presence of disulfide bonds is not yet known. As a preliminary study, the inventors used dissection in BI-1 with HT1080 cells (4) transfected with purified recombinant BI-1 and pcDNA3-BI-1 using diagonal electrophoresis (11) and subsequent immunoblotting. Attempts were made to identify the disulfide bridge. However, disulfide bonds were not detected (results not shown). Therefore, further studies have to be conducted to characterize the structural features of BI-1, but the present inventors were able to avoid refolding problems that could be caused by the formation of disulfide bonds in the protein.

References

[1] Q. Xu, J.C. Reed, Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast, Mol. Cell 1 (1998) 337-346.

[2] H.J. Chae, N. Ke, H. R. Kim, S. Chen, A. Godzik, M. Dickman, J.C. Reed, Evolutionarily conserved cytoprotection provided by Bax inhibitor-1 homologs from animals, plants, and yeast, Gene 323 (2003) 101-113.

[3] H.J. Chae, H. R. Kim, C. Xu, B. Bailly-Maitre, M. Krajewska, S. Krajewski, S. Banares, J. Cui, M. Digicaylioglu, N. Ke, S. Kitada, E. Monosov, M. Thomas, C.L. Kress, J.R. Babendure, R.Y. Tsien, S.A. Lipton, J. C. Reed, BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress, Mol. Cell 15 (2004) 355-366.

[4] HR Kim, GH Lee, KC Ha, T. Ahn, JY Moon, BJ Lee, SG Cho, S. Kim, YR Seo, YJ Shin, JC Reed, HJ Chae, Bax inhibitor-1 is a pH-dependent regulator of Ca ²⁺ channel activity in the endoplasmic reticulum, J. Biol. Chem. 283 (2008) 15946-15955.

[5] V.E. Vaskovsky, E.Y. Kostetsky, I.M. Vasendin, A universal reagent for phospholipid analysis, J. Chromatogr. 114 (1975) 129-141.

[6] C.K. Kim, H.S. Chung, M.K. Lee, L.N. Choi, M.H. Kim, Development of dried liposomes containing ..- galactosidase for the digestion of lactose in milk. Int. J. Pharm. 183 (1999) 185-193.

[7] M. Reichlin, Use of glutaraldehyde as a coupling agent for proteins and peptides. Methods Enzymol. 70 (1980) 159-165.

[8] S. Kolchens, V. Ramaswami, J. Birgenheier, L. Nett, D.F. O .. Brien, Quasi-elastic light scattering determination of the size distribution of extruded vesicles, Chem. Phys. Lipids 65 (1993) 1-10.

[9] R. Hukelhoven, Bax inhibitor-1, an ancient cell death suppressor in animals and plants with prokaryotic relatives, Apoptosis 9 (2004) 299-307.

[10] N. Greenfield, G.D. Fasman, Computed circular dichroism spectra for the evaluation of protein conformation, Biochemistry 8 (1969) 4108-4116.

[11] J.R. Brown, B.S. Hartley, Location of disulphide bridges by diagonal paper electrophoresis, Biochem. J. 101 (1966) 214-228.

Figure 1. Preparation and Reconstitution of BI-1. (a) Inclusions dissolved in urea and CHAPS (lanes 1, using about 7 μg and 0.05 μg protein for SDS-PAGE and Western blotting, respectively) and reconstituted samples (lane 2, SDS-PAGE and Western blots) SDS-PAGE and Western blotting of BI-1 were performed with about 2 μg and 0.05 μg protein, respectively, for the loting. Antibodies against the C-terminal region of BI-1 were used. WB stands for Western Blotting. (b) Phospholipid concentration-dependent reconstitution of BI-1. After preparation of the proteoliposomes, the amount of BI-1 in the liposomes was determined.

Figure 2. Acidic pH-induced Ca ²⁺ release from BI-1 reconstituted into lipid vesicles. (a) After rapid dilution of the proteoliposome with an acidic solution of pH 5.5, the change in fluorescence intensity of indo-1 was recorded kinnetically. (au means arbitrary unit). (b) the amount of Ca ²⁺ released by the acidic pH is expressed as a percentage of the maximum releaseable Ca ²⁺ using fluorescence and radioactivity of ⁴⁵ Ca ²⁺ , and added to the addition of 1% Triton X-100 (control) Compared to that caused by (set to 100%). Data shown are mean ± standard error (SE) of 5 independent experiments.

Figure 3. CD spectrum of BI-1 reconstituted with lipid vesicles. BI-1 (0.8 μM) in liposomes was suspended in buffer solutions with each indicated pH and CD spectra were recorded. Mean residue ellipticity, [θ] _R, is expressed based on the number of amino acids per molecule of BI-1.

<110> Chonnam Industry universtiy Cooperaton Foundation <120> Refolding and reconstitution of human recombinant Bax inhibitor-1          into liposomes from inclusion bodies expressed in Escherichia          coli <130> PN082935 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 237 <212> PRT <213> Homo sapiens <400> 1 Met Asn Ile Phe Asp Arg Lys Ile Asn Phe Asp Ala Leu Leu Lys Phe   1 5 10 15 Ser His Ile Thr Pro Ser Thr Gln Gln His Leu Lys Lys Val Tyr Ala              20 25 30 Ser Phe Ala Leu Cys Met Phe Val Ala Ala Ala Gly Ala Tyr Val His          35 40 45 Met Val Thr His Phe Ile Gln Ala Gly Leu Leu Ser Ala Leu Gly Ser      50 55 60 Leu Ile Leu Met Ile Trp Leu Met Ala Thr Pro His Ser His Glu Thr  65 70 75 80 Glu Gln Lys Arg Leu Gly Leu Leu Ala Gly Phe Ala Phe Leu Thr Gly                  85 90 95 Val Gly Leu Gly Pro Ala Leu Glu Phe Cys Ile Ala Val Asn Pro Ser             100 105 110 Ile Leu Pro Thr Ala Phe Met Gly Thr Ala Met Ile Phe Thr Cys Phe         115 120 125 Thr Leu Ser Ala Leu Tyr Ala Arg Arg Arg Ser Tyr Leu Phe Leu Gly     130 135 140 Gly Ile Leu Met Ser Ala Leu Ser Leu Leu Leu Leu Ser Ser Leu Gly 145 150 155 160 Asn Val Phe Phe Gly Ser Ile Trp Leu Phe Gln Ala Asn Leu Tyr Val                 165 170 175 Gly Leu Val Val Met Cys Gly Phe Val Leu Phe Asp Thr Gln Leu Ile             180 185 190 Ile Glu Lys Ala Glu His Gly Asp Gln Asp Tyr Ile Trp His Cys Ile         195 200 205 Asp Leu Phe Leu Asp Phe Ile Thr Val Phe Arg Lys Leu Met Met Ile     210 215 220 Leu Ala Met Asn Glu Lys Asp Lys Lys Lys Glu Lys Lys 225 230 235 <210> 2 <211> 711 <212> DNA <213> Homo sapiens <400> 2 atgaacatat ttgatcgaaa gatcaacttt gatgcgcttt taaaattttc tcatataacc 60 ccgtcaacgc agcagcacct gaagaaggtc tatgcaagtt ttgccctttg tatgtttgtg 120 gcggctgcag gggcctatgt ccatatggtc actcatttca ttcaggctgg cctgctgtct 180 gccttgggct ccctgatatt gatgatttgg ctgatggcaa cacctcatag ccatgaaact 240 gaacagaaaa gactgggact tcttgctgga tttgcattcc ttacaggagt tggcctgggc 300 cctgccctgg agttttgtat tgctgtcaac cccagcatcc ttcccactgc tttcatgggc 360 acggcaatga tctttacctg cttcaccctc agtgcactct atgccaggcg ccgtagctac 420 ctctttctgg gaggtatctt gatgtcagcc ctgagcttgt tgcttttgtc ttccctgggg 480 aatgttttct ttggatccat ttggcttttc caggcaaacc tgtatgtggg actggtggtc 540 atgtgtggct tcgtcctttt tgatactcaa ctcattattg aaaaggccga acatggagat 600 caagattata tctggcactg cattgatctc ttcttagatt tcattactgt cttcagaaaa 660 ctcatgatga tcctggccat gaatgaaaag gataagaaga aagagaagaa a 711 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gacggatcca tgaacatatt tgatcgaaag 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gacaagcttt tatttcttct ctttcttctt 30 <210> 5 <211> 13 <212> PRT <213> Homo sapiens <400> 5 Cys Ala Met Asn Glu Lys Asp Lys Lys Lys Glu Lys Lys   1 5 10  

Claims (20)

Isolating an inclusion body comprising the BI-1 protein from the prokaryotic cell culture; Dissolving the isolated inclusion body in a first buffer comprising a first concentration of chaotropic agent, a reducing agent and an amphoteric surfactant; Mixing the first buffer solution with phospholipids; Exchanging a first buffer in the mixture for a second buffer, the second buffer comprising a chaotropic agent at a second concentration lower than the first concentration, and the reducing agent and no amphoteric surfactant Not; Exchanging the second buffer solution with a third buffer, wherein the third buffer comprises the reducing agent and does not include the chaotropic agent and the amphoteric surfactant; And Separating the liposomes comprising the BI-1 protein from the third buffer solution; the method of producing a liposome comprising the BI-1 protein. The method of claim 1, wherein the chaotropic agent is urea or guanidinium chloride. The method of claim 1, wherein the reducing agent is selected from the group consisting of DTT (dithiothritol), cysteine, 2-mercapto ethanol, and TCEP (tris (2-carboxyethyl) phosphine). The method of claim 1, wherein the amphoteric surfactant is selected from the group consisting of CHAPS, CHAPSO, cholate, and deoxycholate. The method of claim 1, wherein the first concentration of chaotropic agent is 6M to 8M urea or at least 6M guanidinium chloride. The method of claim 1, wherein the first buffer is a buffer comprising 8M urea, 0.1 mM DTT and 1.5% (w / v) CHAPS. The method of claim 1, wherein the first buffer is a HEPES buffer, Tris-HCl buffer or phosphate buffer. The method of claim 1, wherein the first buffer further comprises CaCl ₂ . The method of claim 1, wherein the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The method of claim 1, wherein the second concentration of chaotropic agent is 1.5M to 5M urea or 1M to 4M guanidinium glolide. The method of claim 1, wherein the first buffer is a buffer comprising 20 mM HEPES buffer (pH 7.4), 8M urea, 0.1 mM DTT, 1 mM CaCl ₂ and 1.5% (w / v) CHAPS. The method of claim 1, wherein the second buffer is a buffer comprising 20mM HEPES buffer (pH 7.4), 2M urea, 0.1mM DTT and 1mM CaCl ₂ . The method of claim 1, wherein the third buffer is a buffer comprising 20mM HEPES buffer (pH 7.4), 0.1mM DTT and 1mM CaCl ₂ . The method of claim 1, wherein the exchange is performed by dialysis or ultradialysis. The method of claim 1, further comprising washing the separated liposomes. The method of claim 1, further comprising washing with a fourth buffer comprising 20 mM HEPES buffer, pH 7.4, 100 mM NaCl, 0.1 mM DTT, 0.5 mM EGTA, and 1 M KCl. The method of claim 16, further comprising exchanging said fourth buffer solution with a fifth buffer from which 0.5 mM EGTA and 1M KCl have been removed from said fourth buffer. 18. The method of claim 17, wherein the exchange is performed by dialysis or ultradialysis. The method of claim 1, further comprising performing cation exchange chromatography on the isolated liposomes. A liposome comprising a BI-1 protein prepared by the method according to any one of claims 1 to 19.

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