Polypeptide cleavage method using OmpT protease variant
09657323 ยท 2017-05-23
Assignee
Inventors
Cpc classification
C12P21/06
CHEMISTRY; METALLURGY
International classification
Abstract
A polypeptide cleavage method characterized in that arginine or lysine is at the P1 position of a desired cleavage site in a polypeptide, an amino acid other than aspartic acid, glutamic acid or proline is at the P1 position, a single basic amino acid or two or three consecutive basic amino acids are situated at any site in the amino acid sequence from the P10 position to the P3 position or from the P3 position to the P5 position (with the proviso that a single basic amino acid is not situated at the P6 or P4 position), and OmpT protease or its variant enzyme having a substitution at the 97th amino acid from the N-terminus is used to cleave the desired cleavage site in the polypeptide.
Claims
1. A process for cleaving a polypeptide comprising cleaving the polypeptide with an E. coli OmpT protease variant consisting of an amino acid substitution at the 97th position of the amino acid sequence of SEQ ID NO: 41, wherein the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine, methionine, or histidine, wherein the polypeptide comprises a cleavage site that is a peptide bond between the P1 position and the P1 position, and wherein the P1 position is arginine or lysine and the P1 position is: (1) serine or alanine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine; (2) phenylalanine, alanine, serine, cysteine, or tyrosine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is methionine; or (3) alanine, valine, isoleucine, methionine, serine, threonine, cysteine, or asparagine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is histidine.
2. The process of claim 1, wherein the amino acid sequence from the P10 position to the P3 position comprises only a single basic amino acid or only two or three consecutive basic amino acids.
3. The process of claim 2, wherein the basic amino acids are arginine and/or lysine.
4. The process of claim 3, wherein the basic amino acids are arginine.
5. The process of claim 1, wherein the polypeptide is a fusion protein comprising a protecting peptide and a target peptide, wherein the C-terminus of the protecting peptide is the P1 position and the N-terminus of the target peptide is the P1 position, wherein the fusion protein is produced by expressing a gene encoding the fusion protein in a host cell, and wherein cleavage of the fusion protein liberates the target peptide.
6. The process of claim 5, wherein a single basic amino acid or two or three consecutive basic amino acids are situated in the amino acid sequence from the P10 position to the P3 position.
7. The process of claim 5, wherein the E. coli OmpT protease variant is produced by expressing a gene encoding the E. coli OmpT protease variant in said host cell.
8. The process of claim 1, wherein two or three consecutive basic amino acids are situated between the P10 and P3 positions in the polypeptide.
9. The process of claim 8, wherein three consecutive basic amino acids are situated between the P5 and P3 positions in the polypeptide.
10. A process for cleaving a polypeptide comprising cleaving the polypeptide with an E. coli OmpT protease variant consisting of an amino acid substitution at the position of the amino acid sequence of SEQ ID NO: 41, wherein the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine, methionine, or histidine, wherein the polypeptide comprises a cleavage site that is a peptide bond between the P1 position and the P1 position, and wherein the P1 position is arginine or lysine and the P1 position is: (1) serine or alanine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine; (2) phenylalanine, alanine, serine, cysteine, or tyrosine when the 97th amino acid from the N-terminus of the E.coli OmpT protease variant is methionine: or (3) alanine, valine, isoleucine, methionine, serine, threonine, cysteine, or asparagine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is histidine, and wherein the amino acid sequence from the P5 to P1 positions in the polypeptide is Arg-Arg-Arg-Ala-Arg (SEQ ID NO: 11).
11. A process for cleaving a polypeptide comprising cleaving the polypeptide with an E. coli OmpT protease variant consisting of an amino acid substitution at the 97the position of the amino acid sequence of SEQ ID NO: 41, wherein the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine, methionine, or histidine, wherein the polypeptide comprises a cleavage site that is a peptide bond between the P1 position and the P1 position, and wherein the P1 position is: (1) serine or alanine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is leucine; (2) phenylalanine, alanine, serine, cysteine, or tyrosine when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is methionine; or (3) alanine, valine, isoleucine, methionine, serine threonine, cysteine, or asparagine, when the 97th amino acid from the N-terminus of the E. coli OmpT protease variant is histidine, and wherein the amino acid sequence from the P7 to P1 positions in the polypeptide is Asp-Ala-Arg-Arg-Arg-Ala-Arg (SEQ ID NO: 12).
12. The process of claim 5, wherein the target peptide consists of between 22 and 45 amino acid residues.
13. The process of claim 12, wherein the target peptide is adrenocorticotropic hormone (1-24), motilin, or calcitonin precursor.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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BEST MODE FOR CARRYING OUT THE INVENTION
(19) The present invention will now be explained in greater detail.
(20) Plasmid pG117S4HompPRR is an expression plasmid which expresses a fusion protein (PRR) comprising glucagon-like peptide-1 (7-37) (GLP-1(7-37)).
(21) The protected protein of this fusion protein is composed of -gal117S4H containing the 117 N-terminal amino acids of E. coli -galactosidase as the protecting protein, a linker sequence comprising 26 amino acids containing an arginine-arginine sequence, and GLP-1(7-37). The present inventors had already discovered that E. coli OmpT protease (SEQ ID NO: 41) cleaves the central peptide bond of the arginine-arginine sequence in the PRR linker sequence, releasing a target polypeptide of 44 amino acids containing GLP-1(7-37) (Okuno, K. et al. Biosci., Biotechnol. Biochem. 66:127-134, 2002).
(22) The present inventors constructed PA based on the fusion protein (PRR), as a fusion protein having arginine at the P1 and P1 positions and having all of the other amino acids from the P10 to P5 positions replaced with alanine.
(23) Also, a fusion protein (PAn) was constructed starting from the fusion protein PA and replacing the alanine at each position with arginine, and the effect on OmpT protease cleavage by situating the basic amino acid arginine adjacent to the OmpT protease cleavage site was examined.
(24) As a result, it was newly discovered that the cleavage efficiency can be increased if a basic amino acid (for example, arginine or lysine) is present at positions between P10 and P3 or positions P3 and P5 of the amino acid sequence adjacent to the cleavage site (except for cases where only position P6 or P4 is replaced with a basic amino acid).
(25) On the other hand, when P2 or P2 is arginine, the sequence contains three consecutive arginines instead of only the two arginines at positions P1 and P1 situated at the cleavage site, and this case was found instead to have a reduced cleavage efficiency. That is, although the cleavage efficiency is increased if arginine is present around the cleavage site, the cleavage efficiency is reduced in the case of three consecutive arginines, and therefore the cleavage efficiency can be controlled by substituting arginine in the cleavage site adjacent amino acid sequence.
(26) For fusion protein PA3 wherein arginine is present at position P3 (cleavage site adjacent amino acid sequence=-Ala-Ala-Arg[P1]-Arg[P1]-Ala-Arg[P3]-Ala[P4]-Ala-) (SEQ ID NO: 14), it was found that cleavage also occurs between arginine at position P3 and alanine at position P4, and a sequence was discovered that allowed efficient cleavage at the arginine-alanine site. Since the fact that the substrate was efficiently cleaved with a sequence other than one having consecutive basic amino acids is extremely important for using OmpT protease as a processing enzyme, the present inventors carried out further investigation.
(27) Upon investigating various amino acid sequences based on the knowledge that cleavage efficiency is increased by situating arginine at the cleavage site adjacency and the knowledge that three consecutive arginines render cleavage between arginine-arginine more difficult, it was found that in the amino acid sequence -Arg-Arg-Arg-Ala-Arg-Ala- (SEQ ID NO: 15), the major cleavage occurs at -Arg-Arg-Arg-Ala-ArgsAla-(SEQ ID NO: 15). In other words, this demonstrated a property whereby situating three consecutive basic amino acids promotes cleavage at basic amino acid sites thereafter.
(28) However, cleavage did occur in the aforementioned amino acid sequence (-Arg-Arg-Arg-Ala-Arg-Ala-) (SEQ ID NO: 15) even in the three consecutive arginine residue sequence. In order to inhibit this, the amino acid sequence -Asp-Ala-Arg-Arg-Arg-Ala-ArgLAla- (SEQ ID NO: 16) was constructed having aspartic acid situated as an acidic amino acid in the amino acid sequence upstream from the N-terminal end. The arginine-alanine cleavage efficiency was reduced by half using this sequence, but cleavage in the three consecutive arginine sequence was successfully inhibited. That is, cleavage by OmpT protease may be optimized for easier cleavage between arginine-alanine in -Arg-Arg-Arg-Ala-ArgAla- (SEQ ID NO: 15) and Asp-Ala-Arg-Arg-Arg-Ala-ArgsAla- (SEQ ID NO: 16). It was thought that using these sequences (-Arg-Arg-Arg-Ala-Arg-Ala- (SEQ ID NO: 15) and Asp-Ala-Arg-Arg-Arg-Ala-Arg-Ala- (SEQ ID NO: 16)), and most preferably Asp-Ala-Arg-Arg-Arg-Ala-Arg-Ala- (SEQ ID NO: 16), would allow efficient cleavage even when the P1 position is an amino acid other than alanine.
(29) Based on these results, motilin (with phenylalanine as the N-terminal amino acid) was examined as a target polypeptide to determine whether or not situating a physiologically active peptide in the amino acid sequence at the C-terminal end of the cleavage site -Arg-Arg-Arg-Ala-ArglAla- (SEQ ID NO: 15) permits direct cleavage of the physiologically active peptide from a fusion protein with OmpT protease. Fusion protein PMT was constructed with motilin as the target polypeptide, and was reacted with OmpT protease in an attempt to cut off motilin.
(30) However, it was shown that motilin is not efficiently cut off from the fusion protein PMT. This result suggested that, while the substrate specificity of OmpT protease is known to be tolerant with regard to the amino acid at position P1, more efficient cleavage requires introduction of a mutation into the protease itself to increase the specificity for the amino acid at position P1.
(31) Literature analyzing the crystal structure of OmpT has already been published (Vandeputte-Rutten, L. et al. EMBO J. 20: 5033-5039, 2001), and a related report (Kramer, R A. et al. FEBS Lett. 505: 426-430, 2001) suggests possible interaction between the P1 position amino acid of the substrate and the Asp.sup.97 (97th position aspartic acid from the N-terminus) of OmpT protease. In order to investigate the change in substrate specificity that results by substitution of an amino acid at position 97, a plasmid was created for a mutant having Asp.sup.97 of OmpT replaced with the 20 different amino acids (including synonymous substitution to aspartic acid), and these were introduced into OmpT-deficient E. coli BL21 to prepare 20 E. coli strains expressing the OmpT protease variants OmpT D97X (where X corresponds to the 20 amino acids).
(32) In order to examine the P1 position substrate specificity of OmpT protease, these were reacted with the fusion protein PRX (where X corresponds to the 20 amino acids, see Japanese Patent Application No. 2000-602803) having the structure shown in
(33) Based on these findings, and considering that the N-terminal amino acid of motilin is phenylalanine, the aforementioned fusion protein PMT was reacted with OmpT D97M which exhibited good cleavage for phenylalanine at the P1 position, whereby it was possible to efficiently cut off motilin. That is, by optimizing the sequence adjacent to the cleavage site of OmpT protease and utilizing an OmpT protease variant, the present inventors succeeded in achieving cleavage with OmpT protease with which cleavage at desired sites has been difficult.
(34) Also, in order to verify that the method is industrially applicable, fusion protein PMT-expressing E. coli and OmpT D97M protease variant-expressing E. coli were cultured at high density and the OmpT D97M protease variant-expressing E. coli itself was directly added to a reaction solution containing inclusion bodies prepared from the fusion protein PMT-expressing E. coli, and allowed to react at 25 C. for 1 hour. After adding 20 mM acetic acid (pH 4.0) to the reaction solution and removing the precipitate, the supernatant was supplied for cation-exchange and reverse-phase chromatography. This procedure allowed production of 160 mg per liter of fusion protein PMT-expressing E. coli culture solution, at a yield of 52% motilin with a purity of 99.0% or greater, which is an industrially acceptable level.
(35) In order to confirm the general utility of this polypeptide production system, a fusion protein was prepared comprising human adrenocorticotropic hormone(1-24) (N-terminal amino acid: serine) as the target polypeptide and human calcitonin precursor (N-terminal amino acid: cysteine), and was treated with the OmpT protease variant. As a result, it was possible to obtain the desired target polypeptide in all cases, thus demonstrating the general utility of the system.
(36) E. coli co-expressing the fusion protein PMT and OmpT D97M protease variant was prepared, and it was confirmed that human motilin can be released from the fusion protein PMT by simple dissolution in urea of inclusion bodies obtained by culturing the E. coli.
(37) The specific experimental procedures not described in the examples provided below were as follows, unless otherwise specified.
(38) (1) Construction of Expression Plasmids
(39) The expression plasmids were constructed by an ordinary protocol using E. coli JM109. Identity of the constructed expression plasmid as the target plasmid was confirmed by DNA sequence determination of the DNA region obtained by PCR for mutation introduction and the DNA region obtained by substitution with synthetic DNA. The structures of the plasmids constructed for Examples 1, 3, 5, 7, 9, 16 and 18 are shown in
(40) (2) Assay of OmpT Protease Enzyme Activity
(41) The OmpT protease activity was assayed using dynorphin A (Peptide Research Laboratory) as the substrate.
(42) After adding 5 L of 1 mg/mL dynorphin A to 40 L of 50 mM sodium phosphate (pH 6.0) containing 0.1% Triton X-100, a 5 L OmpT protease activity assay sample was added thereto and reaction was initiated. The reaction was performed at 25 C. for 10 minutes and terminated by addition of 5 L of 1N HCl. The reaction solution was centrifuged at 10,000g for 3 minutes, the supernatant was recovered, and 20 L thereof was supplied for HPLC analysis.
(43) The HPLC analysis was carried out using a YMC PROTEIN RP column, with a column temperature of 40 C. and a flow rate of 1 mL/min. After rinsing with 10% acetonitrile containing 0.1% trifluoroacetic acid for 3 minutes, elution was performed with a linear gradient of 10-15% acetonitrile containing 0.1% trifluoroacetic acid for 10 minutes. Absorption at 220 nm was monitored, and the decomposition product peptide Tyr-Gly-Gly-Phe-Leu-Arg (SEQ ID NO: 17) was detected. The OmpT protease activity upon cleavage of 1 mol dynorphin A at 25 C. for 1 minute was defined as 1 unit.
(44) (3) SDS-polyacrylamide Electrophoresis
(45) The SDS-polyacrylamide electrophoresis used to investigate cleavage of the fusion protein employed 16% Peptide-PAGEmini by Tefco as the gel, Tricine electrophoresis buffer by Biorad as the electrophoresis buffer, and a protein molecular weight marker by Tefco or Biorad as the molecular weight marker. An equivalent of 2SDS-PAGE sample buffer containing 4 M urea was added to the sample prior to heating at 100 C. for 5 minutes. A 10 L portion was supplied for electrophoresis, and electrophoresis was carried out under the electrophoresis conditions indicated by Tefco. After electrophoresis, dyeing was performed with a dyeing solution containing Coumassie Brilliant Blue R-250.
(46) (4) Preparation of Inclusion Bodies
(47) In the examples, the fusion proteins were expressed as inclusion bodies in E. coli, and cleavage by OmpT protease occurs simply by dissolution of the obtained inclusion bodies in urea if the E. coli also express OmpT protease. In order to avoid cleavage, therefore, the OmpT protease-deficient E. coli strain W3110 M25 was transformed with the fusion protein-expressing plasmid, and each fusion protein was expressed as inclusion bodies. The W3110 M25 recombinant E. coli expressing each fusion protein was subjected to gyratory culture at 150 rpm, 37 C. overnight using 400 mL of LB liquid medium (0.5% (w/v) yeast extract, 1% (w/v) tryptone, 0.5% sodium chloride) containing 10 mg/L tetracycline in a 2 L Erlenmeyer flask.
(48) On the following day, the cells were recovered by centrifugation at 4 C., 6000g for 10 minutes, and then subjected to ultrasonication for cell disruption. Deionized water was added to the disrupted cells to 30 mL, and after centrifugation at 4 C., 25,000g for 15 minutes the supernatant was discarded and the precipitated fraction (of inclusion bodies) was recovered. This was suspended in 30 mL of 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1% Triton X-100 and centrifuged at 4 C., 25,000g for 15 minutes to obtain a precipitate. The precipitate was suspended in 30 mL of deionized water and centrifuged at 4 C., 25,000g for 15 minutes, and the precipitate was recovered. Deionized water was added thereto to 1.5 mL and the obtained suspension was centrifuged at 4 C., 10,000g for 30 minutes to obtain a precipitate, and after repeating this procedure, the precipitate was suspended in deionized water to OD.sub.660=100; the inclusion bodies prepared in this manner were used as substrate for OmpT protease reaction.
(49) (5) OmpT Protease Reaction
(50) OmpT protease reaction using the fusion protein as substrate was performed in the following manner. After adding 2.5 L of 1 M sodium phosphate (pH 7.0) and 2 L of 50 ml EDTA to 20 L of 20 M urea, 10 L of fusion protein inclusion bodies (OD.sub.660=100) was added for lysis of the inclusion bodies. There was then added 10.5 L of water, followed by 5 L of 1.4 units/mL OmpT protease, and reaction was initiated with a reaction mixture volume of 50 L. The reaction temperature was 25 C. and reaction was performed for 30 minutes.
(51) Quantitation of the polypeptide obtained by reaction with OmpT protease was accomplished by HPLC under the following conditions, unless otherwise specified. The reaction was terminated by addition of 150 L of 6% acetic acid, 2 M urea to the OmpT protease reaction mixture, and upon centrifugation at 10,000g for 3 minutes, 20 l of the supernatant was supplied to a YMC PROTEIN RP column. HPLC was carried out at a column temperature of 40 C. and a flow rate of 1 mL/min. Elution was performed with a linear gradient of 30-50% acetonitrile containing 0.1% trifluoroacetic acid for 20 minutes. Absorption at 214 nm was monitored for quantitation of the polypeptide.
(52) (6) Mass Analysis of Polypeptide
(53) In order to presume the cleavage site location, mass analysis of the polypeptide isolated by HPLC was carried out using SSQ710 by Thermo Finnigan.
(54) (7) Preparation of E. coli Outer Membrane Fraction
(55) An outer membrane fraction was prepared in the following manner for E. coli expressing OmpT protease or OmpT protease variant with W3110 M25 as the host cells, and the fraction was used as OmpT protease or OmpT protease variant for fusion protein cleavage reaction in Examples 10, 14, 16 and 18. The culturing method was performed as in (4) above, and upon completion of culturing, cells were obtained by centrifugation at 4 C., 6000g for 10 minutes. The cells were suspended in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (TE), and disrupted by ultrasonication. The disrupted cells were centrifuged at 4 C., 1000g for 10 minutes, the precipitate was discarded and the supernatant was recovered. It was then centrifuged at 4 C., 36,000g for 40 minutes, and the precipitate was recovered, suspended in TE, and again centrifuged at 4 C., 36,000g for 40 minutes. The obtained precipitate was suspended in TE to OD.sub.660=10. It was stored at 20 C. until use.
EXAMPLES
(56) The present invention will now be explained in greater detail through the following examples.
Example 1
Preparation of Fusion Proteins PAn
(57) OmpT protease is an endoprotease found in E. coli outer membrane. Since the basic amino acids in the amino acid sequence adjacent to the cleavage site have a major effect on cleavage by this enzyme, the present inventors utilized the known cleavage site of the enzyme for the experiment described below to investigate the relationship between the basic amino acid position and the cleavage efficiency.
(58) Arginine was substituted for alanine at positions P10 to P2 and positions P2 to P5 of the cleavage site of the fusion protein PA (a fusion protein comprising the protecting protein deriving from 117 amino acids from the N-terminus of E. coli -galactosidase (-gal117S4H) and human glucagon-like peptide-1(7-37) (GLP-1(7-37) via a linker peptide) shown in
(59) Plasmid pG117S4HompPRR (see Japanese Patent Application No. 2000-602803) having the structure shown in
Example 2
Cleavage of Fusion Proteins PAn by OmpT Protease
(60) The cleavage efficiency by OmpT protease was examined using each fusion protein PAn (
(61) All of the PAn proteins underwent cleavage by OmpT protease at the same site as PA, while PA2, PA2 and PA3 also underwent cleavage at other sites (
(62) Increase in cleavage efficiency was seen for all of the PAn proteins except for PA2 and PA2, suggesting that the cleavage efficiency can be improved by situating arginine at positions P10 to P3 and positions P3 to P5 of the amino acid sequence adjacent to the cleavage site. Among these, PA4 had the highest cleavage efficiency of about 5 times that of PA, and therefore substitution of arginine at position P4 was shown to be most effective. On the other hand, the cleavage efficiencies of PA2 and PA2 were reduced to about , indicating that the cleavage efficiency is reduced with a sequence of three consecutive arginines.
Example 3
Preparation of Fusion Proteins PA1A3 PA1A3
(63) OmpT protease is known to be an enzyme which cleaves primarily between consecutive basic amino acids. However, the results of Example 2 demonstrated that the fusion protein PA3 is cleaved at two sites: Arg.sup.140-Arg.sup.141 and Arg.sup.143-Ala.sup.144, and that one of them is -ArgAla-cleavage. The cleavage efficiency at -ArgAla- is low compared to the cleavage efficiency between basic amino acids, but it was speculated that this could be improved to an industrially useful cleavage efficiency.
(64) Therefore, in order to inhibit cleavage at Arg.sup.140-Arg.sup.141 among the two cleavage sites Arg.sup.140-Arg.sup.141 and Arg.sup.143-Ala.sup.144 of the fusion protein PA3, there were prepared fusion proteins PA1A3 and PA1A3 (
(65) The fusion protein PA1A3 and PA1A3 expression plasmids pG117S4HompPA1A3 and pG117S4HompPA1A3 were constructed by introducing base substitutions by PCR into the fusion protein PA3 expression plasmid pG117S4HompPA3, having the structure shown in
Example 4
Cleavage of Fusion Proteins PA1A3 and PA1A3 with OmpT Protease
(66) The cleavage sites and cleavage efficiencies with OmpT protease were investigated for the fusion proteins PA1A3 and PA1A3 shown in
(67) However, all of the cleavage efficiencies were lower than the cleavage efficiency at Arg.sup.140-Arg.sup.141 of PA. Cleavage was also confirmed at Arg.sup.140-Ala.sup.141 in PA1A3. If Arg.sup.143-Ala.sup.144 is considered as the cleavage site P1-P1, this suggests that cleavage at Arg.sup.143-Ala.sup.144 occurs so long as arginine is present at position P3 (Arg.sup.141 in PA1A3) or arginine is present at position P4 (Arg.sup.140 in PA1A3), but that PA3 with arginine situated at both positions P4 and P3 has a higher cleavage efficiency than PA1A3 and PA1A3.
Example 5
Preparation of Fusion Proteins PA23 PA323 and PA23
(68) The results of Example 4 demonstrated that the fusion proteins PA1A3 and PA1A3 are cleaved at Arg.sup.143-Ala.sup.144, and notably only at Arg.sup.143-Ala.sup.144 in PA1A3, but the cleavage efficiencies were low. Thus, amino acid substitutions were introduced into the fusion protein PA3 in order to design an amino acid sequence with an increased cleavage efficiency at -ArgAla- (Arg.sup.143-Ala.sup.144). Based on the results of Example 2, the fusion proteins PA23, PA323 and PA23 (
(69) The fusion protein PA23 and PA23 expression plasmids pG117S4HompPA23 and pG117S4HompPA23 were constructed by introducing base substitutions by PCR into the fusion protein PA3 expression plasmid pG117S4HompPA3 having the structure shown in
Example 6
Cleavage of Fusion Proteins PA23, PA323 and PA23 with OmpT Protease
(70) The cleavage sites and cleavage efficiencies with OmpT protease were investigated for the fusion proteins PA23, PA323 and PA23 shown in
(71) Cleavage was also observed at Arg.sup.139-Arg.sup.140 and Arg.sup.140-Arg.sup.141, but was 13% of the cleavage efficiency at Arg.sup.143-Ala.sup.144. The cleavage efficiency at Arg.sup.143-Ala.sup.144 of PA323 was also 2.9 times the cleavage efficiency at Arg.sup.140-Arg.sup.141 of PA, but cleavage was also observed at Arg.sup.140-Arg.sup.141, at 59% the cleavage efficiency at Arg.sup.143-Ala.sup.144. In PA23, the cleavage efficiency at Arg.sup.143-Ala.sup.144 was low at 63% of the cleavage efficiency at Arg.sup.140-Arg.sup.141 of PA, and cleavage at Arg.sup.140-Arg and Arg.sup.142-Arg.sup.143 was also confirmed. This indicated that among these three fusion proteins, PA23 has the optimum sequence for increasing the cleavage efficiency at -ArgAla- (Arg.sup.143-Ala.sup.144) and decreasing the cleavage efficiency between consecutive basic amino acids.
Example 7
Preparation of Fusion Proteins PA5D23 PA4D23 and PA3D23
(72) The results of Example 6 demonstrated that the cleavage efficiency at Arg.sup.143-Ala.sup.144 in the fusion protein PA23 is very high. However, cleavage was also confirmed at Arg.sup.139-Arg.sup.140 and Arg.sup.140-Arg.sup.141. Thus, since cleavage is presumably inhibited when acidic amino acids are present near the cleavage site, there were prepared in the following manner fusion proteins PA5D23, PA4D23 and PA3D23 (
(73) The fusion protein PA5D23, PA4D23 and PA3D23 expression plasmids pG117S4HompPA5D23, pG117S4HompPA4D23 and PG117S4HompPA3D23 were constructed by introducing base substitutions by PCR into the fusion protein PA23 expression plasmid pG117S4HompPA23, having the structure shown in
Example 8
Cleavage of Fusion Proteins PA5D23 PA4D23 and PA3D23 with OmpT Protease
(74) The cleavage sites and cleavage efficiencies with OmpT protease were investigated for the fusion proteins PA5D23, PA4D23 and PA3D23 shown in
(75) In particular, the cleavage efficiency at Arg.sup.143-Ala.sup.144 of PA4D23 was low compared to PA23, but twice the cleavage efficiency at Arg.sup.140-Arg.sup.141 of PA. On the other hand, no cleavage was detected at Arg.sup.139-Arg.sup.140 and Arg.sup.140-Arg.sup.141 as was detected with PA23. That is, if Arg.sup.140-Arg.sup.141 is considered as P1-P1, its cleavage was likely inhibited by the aspartic acid at position P3. Similarly, if Arg.sup.139-Arg.sup.140 is considered as P1-P1, its cleavage was likely inhibited by the aspartic acid at position P2. The cleavage efficiency at Arg.sup.143-Ala.sup.144 of PA5D23 was also 1.9 times the cleavage efficiency at Arg.sup.140-Arg.sup.141 of PA, but cleavage was also observed at Arg.sup.140-Arg.sup.141.
(76) For PA3D23, no cleavage was detected at Arg.sup.139-Arg.sup.140 and Arg.sup.140-Arg.sup.141, similar to PA4D23. That is, if Arg.sup.140-Arg.sup.141 is considered as P1-P1, its cleavage was likely inhibited by the aspartic acid at position P4. Similarly, if Arg.sup.139-Arg.sup.140 is considered as P1-P1, its cleavage was likely inhibited by the aspartic acid at position P3. However, the cleavage efficiency at Arg.sup.143-Ala.sup.144 was about the same as the cleavage efficiency at Arg.sup.140-Arg.sup.141 of PA, and lower than PA4D23. This indicated that among these three fusion proteins, PA4D23 has the optimum sequence for increasing the cleavage efficiency at -ArgAla- (Arg.sup.143-Ala.sup.144) and decreasing the cleavage efficiency between consecutive basic amino acids.
(77) This suggests the possibility that when OmpT protease is used to cut off a target polypeptide, wherein the N-terminal amino acid is any of the 17 amino acids other than aspartic acid, glutamine and proline, from a fusion protein having the structure: protecting protein-linker peptide-target polypeptide, specific cleavage is possible by situating the target polypeptide after the C-terminal of the amino acid sequence -Asp-Ala-Arg-Arg-Arg-Ala-Arg- (SEQ ID NO: 12).
Example 9
Preparation of Fusion Proteins PRMT and PMT
(78) The results in Example 6 indicated that OmpT protease can efficiently cleave -ArgAla- in the amino acid sequence adjacent to the cleavage site of this enzyme in the fusion protein PA23 shown in
(79) First, fusion protein PRMT (
(80) The structures of the fusion protein PRMT expression plasmid pG117S4HompPRMT and the PMT expression plasmid pG117S4HompPMT are shown in
Example 10
Cleavage of Fusion Proteins PRMT and PMT by OmpT Protease
(81) Cleavage of the fusion proteins PRMT and PMT shown in
(82) This demonstrated that human motilin cannot be cut off at the primary peptide cleavage site by OmpT protease, simply by using -Arg-Arg-Arg-Ala-Arg-motilin (SEQ ID NO: 11) as the amino acid sequence adjacent to the cleavage site. It was thus suggested that, while the substrate specificity of this protease is tolerant with regard to the amino acid at position P1, more efficient cleavage requires introduction of a mutation into the protease itself, to increase the specificity for the amino acid at position P1. Thus, an OmpT protease variant was created and it was examined whether or not primarily human motilin can be cut off from the fusion protein using it.
Example 11
Preparation of OmpT protease variant-expressing E. coli
(83) It being noted that literature analyzing the crystal structure of OmpT protease (Vandeputte-Rutten, L. et al. EMBO J. 20: 5033-5039, 2001) and a related report (Kramer, R A. et al. FEBS Lett. 505: 426-430, 2001) suggest possible interaction between the P1 position amino acid of the substrate and Asp.sup.97 of OmpT protease, a plasmid was created having Asp.sup.97 of OmpT protease replaced with the 20 different amino acids (including synonymous substitution to aspartic acid) using PCR in the manner described below, and these were introduced into OmpT-deficient E. coli BL21 to prepare 20 E. coli strains expressing OmpT protease variants.
(84) In order to facilitate introduction of mutations at Asp.sup.97 of OmpT protease and minimize the DNA region amplified by PCR, first there was constructed an OmpT protease-expressing plasmid pOmpTXbaI, having an XbaI restriction endonuclease site introduced by using PCR for substitution of TCT for the AGT coding for Ser.sup.99 of OmpT protease in the OmpT protease-expressing plasmid pOmpTTcE (see Japanese Patent Application No. 2000-602803) which has the structure shown in
(85) The resulting twenty expression plasmids pOmpTD97X were each transferred into OmpT protease-deficient E. coli BL21 to prepare 20 E. coli strains expressing the OmpT protease variants OmpT D97X. The E. coli strains were shake cultured to about OD.sub.660=1 in a test tube at 37 C. using 2 mL of LB broth containing 10 g/mL tetracycline, and then the cells were recovered by centrifugation. Next, 1 mL of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was added thereto for suspension, and the cells were recovered by centrifugation. Also, the same procedure was repeated to obtain cells to which TE was added to OD.sub.660=2 to form suspensions for use as cell suspensions for reaction of the OmpT protease variants OmpT D97X. The cell suspensions were stored at 20 C. until use.
Example 12
Confirmation of OmpT Protease Variant Expression Levels in OmpT Protease Variant-Expressing E. coli Cell Suspensions
(86) In order to confirm that the expression levels of OmpT protease variants in the OmpT protease variant-expressing E. coli cell suspensions were all equivalent in the cell suspensions, anti-OmpT protease antibody was used for Western blotting and immunostaining. The anti-OmpT protease antibody was prepared by immunosensitizing rabbits with purified OmpT protease, purifying the IgG fraction from the antiserum, and recovering from it the fraction with affinity for purified OmpT protease.
(87) The cell suspension corresponding to OD.sub.660=0.01 per lane was supplied for 12% SDS-PAGE, and after completion of electrophoresis, a PVDF membrane was used for Western blotting. The purified transfer membrane was immersed in blocking solution (5% (w/v) skim milk/1TBST*) and shaken for 30 minutes at room temperature. Next, the membrane was immersed in a 1000-fold dilution of anti-OmpT protease antibody in blocking solution, and shaken for 100 minutes at room temperature. The solution was discarded, and washing was performed three times with 1TBST* for 5 minutes. The membrane was then immersed in peroxidase-bound anti-rabbit IgG antibody solution diluted 1000-fold with blocking solution, and shaken for 45 minutes at room temperature.
(88) After washing 4 times with 1TBST* for 10 minutes, detection was performed with an ECL kit (Amersham Pharmacia). No band was detected for the OmpT protease-deficient E. coli BL21 host cells while a band was detected for the other cell suspensions at approximately the same intensity, thus indicating that the expression level of OmpT protease variant in the OmpT protease variant-expressing E. coli cell suspension was probably approximately equal in all of the cell suspensions. (*1TBST=10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 0.05% Tween 20).
Example 13
Investigation of P1 Position Substrate Specificity of OmpT Protease Variants OmpT D97X
(89) Because OmpT protease is present on the E. coli outer membrane, it can be reacted with substrate by simple addition of the cells to the reaction solution. Thus, in order to determine the P1 position substrate specificity of the OmpT protease variants OmpT D97X, the fusion proteins PRX (see Japanese Patent Application No. 2000-602803) having the structures shown in
(90) Next, 10.5 L of water was added thereto, 10 L of the OmpT protease variant-expressing E. coli cell suspension prepared in Example 11 was further added, and reaction was initiated at a reaction solution volume of 50 L. The reaction was performed at 25 C. for 60 minutes. Quantitation of the peptide fragments obtained by the reaction was accomplished by HPLC under the same conditions as for the OmpT protease reaction. The results are shown in Table 1.
(91) TABLE-US-00001 TABLE 1 Cleavage of fusion proteins PRX with OmpT protease variants OmpT D97X Fusion proteins OmpT protease variants OmpT D97X PRX D97D D97A D97L D97F D97M D97S D97T D97C D97N D97Q D97E D97H PRA 5.4 3.8 7.1 3.1 6.0 4.0 6.8 6.2 3.8 4.0 6.5 8.4 PRV 3.5 3.0 3.2 5.0 7.8 PRI 3.1 PRF 4.7 7.7 3.7 4.6 3.4 4.1 PRM 4.6 PRS 3.9 9.1 7.1 7.4 5.6 4.1 4.4 7.2 8.7 PRT 3.0 PRC 3.1 3.9 6.5 3.1 4.6 4.8 4.1 6.9 11 PRY 3.2 6.2 PRN 3.5 PRK 88 3.9 39 4.5 PRR 100 4.0 49 4.6
(92) The cleavage efficiencies are expressed relative to 100% as the cleavage efficiency for reaction between the wild-type OmpT protease (D97D) and the fusion protein PRR. The symbol represents a relative cleavage efficiency of less than 3.0%. The OmpT protease variants D97V, D97I, D97P, D97W, D97G, D97Y, D97K and D97R had relative cleavage efficiencies of less than 3.0% for all of the twenty amino fusion proteins PRX. The fusion proteins PRL, PRP, PRW, PRG, PRQ, PRD, PRE and PRH had relative cleavage efficiencies of less than 3% by all of the OmpT protease variants OmpT D97X.
(93) As a result, it was possible to obtain several variants with relatively high cleavage efficiencies and with different specificities than the wild-type OmpT protease. The highest specificity was exhibited by D97D (wild-type) for fusion proteins PRR and PRK, by D97L for PRS, by D97M for PRF and PRY, and by D97H for PRA, PRV, PRI, PRM, PRT, PRC and PRN. Among these, the D97M variant which had high specificity for PRF was used for reaction with the fusion proteins PRMT and PMT prepared in Example 9, to examine its ability to cut off human motilin.
Example 14
Liberation Human Motilin from Fusion Protein PMT by OmpT Protease D97M Variant
(94) Liberation of human motilin from the human motilin fusion proteins PRMT and PMT (
(95) The reaction was terminated by addition of 150 L of 6% acetic acid, 2 M urea to the reaction solution, and upon centrifugation at 10,000g for 3 minutes, 50 l of the supernatant was supplied to a YMC PROTEIN RP column. HPLC was carried out at a column temperature of 40 C. and a flow rate of 1 mL/min. Elution was performed with a linear gradient of 20-27.5% acetonitrile containing 0.1% trifluoroacetic acid for 15 minutes, and absorption at 214 nm was monitored. The cleavage site was identified by isolation of the polypeptide fragments and mass analysis.
(96) TABLE-US-00002 TABLE 2 Release of motilin from fusion protein PMT by OmpT protease variant OmpT D97M (RRAR disclosed as SEQ ID NO: 13) D97M D97D (wild-type) Fusion protein Motilin* RAR-motilin** RRAR-motilin*** Motilin* RAR-motilin** RRAR-motilin*** PRMT 22 ND PMT 78 ND 24 ND 23 100 ND = Not detected. = Not detectable due to structure of fusion protein. *Release by cleavage at Arg.sup.140-Phe.sup.141 from PRMT, Arg.sup.143-Phe.sup.144 from PMT **Polypeptide comprising Arg-Ala-Arg-motilin released by cleavage at Arg.sup.140-Arg.sup.141 from PMT ***Polypeptide comprising Arg-Arg-Ala-Arg-motilin released by cleavage at Arg.sup.139-Arg.sup.140 from PMT
(97) The cleavage efficiencies are expressed as relative cleavage efficiencies to 100 as the cleavage efficiency at Arg.sup.139-Arg.sup.140 in the case of cleavage of PMT by wild-type D97D. PRMT was not cleaved by D97D wild-type, while cleavage of PMT was primarily cleavage at Arg.sup.139-Arg.sup.140 and Arg.sup.140-Arg.sup.141. When the D97M variant was used, however, PRMT was cleaved and motilin was released. PMT was also cleaved to free motilin, but cleavage was also confirmed at Arg.sup.139-Arg.sup.140. However, the amount of motilin released from PMT was 3.5 times higher than from PRMT. This result indicated that the D97M variant is necessary to cut off motilin, and that the motilin cleavage efficiency varies depending on the sequence adjacent to the cleavage site.
Example 15
Polypeptide Production Example Using OmpT Protease Variant and Motilin as the Model Peptide
(98) As a polypeptide production example using an OmpT protease variant, W3110 M25 motilin fusion protein PMT-producing cells (see Example 9) and OmpT protease variant OmpT D97M-expressing cells (created by transforming W3110 M25 with pOmpTD97M) were each cultured at high density on a 2 L scale, and the W3110 M25/OmpT D97M-expressing cells were used to release motilin from PMT, and then purified to produce motilin. This was accomplished by the following 3 steps.
(99) Quantitation of motilin was accomplished by analyzing the reaction mixture diluted with 6% acetic acid, 2 M urea under the same conditions as the HPLC analysis described in Example 14, using human motilin purchased from Peptide Research Laboratory as the standard sample. The motilin purity was analyzed by HPLC under the same conditions as for quantitation, except that elution was performed with a linear gradient of 0-50% acetonitrile containing 0.1% trifluoroacetic acid for 50 minutes.
(100) (1) 2 L Scale High-Density Culturing of W3110 M25 Motilin Fusion Protein PMT-Producing Strain and OmpT Protease Variant OmpT D97M-Expressing Strain
(101) High-density culturing of W3110 M25 motilin fusion protein PMT-producing cells and OmpT protease variant OmpT D97M-expressing cells was carried out in the following manner, and inclusion bodies and expressing cells were prepared from each. The PMT-producing cells and OmpT D97M-expressing cells were subjected to gyratory culturing overnight in a 500 mL Erlenmeyer flask at 37 C., using 100 mL of LB broth containing 10 mg/L tetracycline. On the following day, it was transferred to a spinner culture vessel containing 2 L of medium comprising 4 g/L K.sub.2HPO.sub.4, 4 g/L KH.sub.2PO.sub.4, 2.7 g/L Na.sub.2HPO.sub.4, 0.2 g/L NH.sub.4Cl, 1.2 g/L (NH.sub.4).sub.2SO.sub.4, 4 g/L yeast extract, 2 g/L MgSO.sub.4.7H.sub.2O, 40 mg/L CaCl.sub.2.2H.sub.2O, 40 mg/L FeSO.sub.4.7H.sub.2O, 10 mg/L MnSO.sub.4.nH.sub.2O, 10 mg/L AlCl.sub.3.6H.sub.2O, 4 mg/L CoCl.sub.2.6H.sub.2O, 2 mg/L ZnSO.sub.4.7H.sub.2O, 2 mg/L Na.sub.2MoO.sub.4.2H.sub.2O, 1 mg/L CuCl.sub.2.2H.sub.2O, 0.5 mg/L H.sub.3BO.sub.4, 15 g/L glucose and 10 mg/L tetracycline, and culturing was initiated at 32 C.
(102) After glucose depletion, glycerol was added to 2% and the culturing temperature was increased to 37 C. Culturing was thereafter continued while subsequently adding glycerol to 2% as it was depleted. The course of culturing is shown in
(103) The precipitate was suspended in 2000 mL of deionized water and centrifuged at 4 C., 6000g for 10 minutes, and the precipitate was recovered. The same procedure was repeated to obtain 26 g of precipitate. This was suspended in 26 mL of deionized water and stored at 20 C. until use as an inclusion body suspension (OD.sub.660-250, 45 mL). The OmpT protease variant W3110 M25/OmpT D97M-expressing cells completed culturing at 20 hours after starting, and the culture volume was 2100 mL. The culture was centrifuged at 4 C., 6000g for 10 minutes to obtain a precipitate. The precipitate was suspended in 2000 mL of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) and centrifuged at 4 C., 6000g for 10 minutes, and the precipitate was recovered. This same procedure was repeated and 311 g of precipitate was recovered. The precipitate was suspended in deionized water and stored at 20 C. until use as a cell suspension (OD.sub.660=320, 390 mL).
(104) (2) Cleavage of Inclusion Body Fusion Protein PMT by OmpT Protease Variant W3110 M25/OmpT D97M-Expressing Cells
(105) After adding 1 mL of 1 M sodium phosphate (pH 7.0) and 0.8 mL of 50 mM EDTA to 8 mL of 10 M urea, 4 mL of fusion protein inclusion bodies PMT (OD.sub.660=250) were added for lysis of the inclusion bodies. Next, 5.2 mL of water was added thereto, 1 mL of the OmpT protease variant W3110 M25/OmpT D97M-expressing cell suspension (OD.sub.660=320) prepared in (1) above was further added, and reaction was initiated at a reaction mixture volume of 20 mL. The reaction was carried out by shaking for 60 minutes at 25 C., 120 min.sup.1. After 60 minutes, 40.5 mL of 20 mM acetic acid (pH 4.0) was added to 13.5 mL of reaction mixture (corresponding to inclusion bodies in 100 mL portion of W3110 M25 motilin fusion protein PMT-producing cell culture), and centrifugation was performed at 4 C., 25,000g for 10 minutes to obtain a supernatant. This procedure eliminated virtually all of the unreacted fusion protein, protecting peptide and E. coli-derived proteins.
(106) Next, 20 mM acetic acid (pH 4.0) was added to the supernatant to a volume of 200 mL, and the mixture was supplied for the following purification. There was then added 20 mM acetic acid (pH 4.0) to the supernatant to lower the pH, in order to allow adsorption in the cation-exchange chromatography described below.
(107) There was detected not only human motilin produced by cleavage at Arg.sup.143-Pbe.sup.144, but also a polypeptide (RRAR-motilin) (SEQ ID NO: 13) produced by cleavage at Arg.sup.139-Arg.sup.140. In SDS-PAGE, RRAR-motilin (SEQ ID NO: 13) was seen with a more concentrated band than human motilin (
(108) (3) Purification of Motilin
(109) A 200 mL portion of the supernatant was applied to SP-Sepharose Fast Flow (27 mL, 26 mm50 mm) by Amersham Pharmacia previously equilibrated with 20 mM acetic acid (pH 4.0). Next, washing was performed by flowing through 20 mM acetic acid (pH 4.0), 20 mM acetic acid (pH 4.0) and 0.1 M NaCl, at 100 mL each. The elution was performed by flowing through 200 mL of 20 mM acetic acid (pH 4.0) at a linear gradient of 0.1-0.5 M NaCl. The flow rate for cation-exchange chromatography was 5 mL/min in all cases. The elution fractions were dispensed in 5 mL portions and based on HPLC analysis results, fractions were selected and pooled. It was thereby possible to remove the polypeptide produced by cleavage of the fusion protein PMT at Arg.sup.139-Arg.sup.140.
(110) The pools were supplied to Vydac 214TPB1520 (24 mL, 10 mm300 mm) which had been previously equilibrated with 0.1% trifluoroacetic acid (TFA). Washing was performed by flowing through 100 mL of 0.1% TFA, and elution was carried out by flowing through 200 mL of 0.1% TFA, with a 0-30% acetonitrile linear gradient. The flow rate for reverse-phase chromatography was 1.6 mL/min in all cases. The eluted fractions were dispensed into 4 mL portions and based on HPLC analysis results, fractions were selected and pooled. The results of the purification are shown in Table 3.
(111) TABLE-US-00003 TABLE 3 Human motilin purification results Human Human Volume motilin motilin Yield Purity Purification stage (mL) (mg/mL) (mg) (%) (%) Reaction 13.5 2.32 31 100 5.87 Acidic precipitate 200 0.159 32 100 48.8 Cation-exchange 40.0 0.591 24 77 94.2 Reverse-phase 22.5 0.696 16 52 >99.0 Purification using inclusion bodies corresponding to 0.1 L W3110 M25 PMT culture solution
(112) The results of HPLC analysis, mass analysis and N-terminal amino acid analysis of the sample obtained by this purification matched those for human motilin. The purification demonstrated that it is possible to obtain 160 mg of human motilin at 99.0% purity at a yield of 52%, per 1 L of W3110 M25 motilin fusion protein PMT-producing cell culture.
Example 16
Physiologically Active Polypeptide Release from Fusion Protein Using OmpT Protease Variant
(113) In order to examine whether or not a physiologically active polypeptide other than human motilin can be released from a fusion protein using an OmpT protease variant, a plasmid was constructed to have the structure shown in
(114) Cleavage fragments of each fusion protein were isolated by HPLC and subjected to mass analysis. The HPLC was carried out using a YMC PROTEIN RP column at a column temperature of 40 C. and a flow rate of 1 mL/min. Elution was performed with a linear gradient of 10-50% acetonitrile containing 0.1% trifluoroacetic acid for 50 minutes, and absorption at 214 nm was monitored. The fusion protein PAC was cleaved at Arg.sup.143-Ser.sup.144 by wild-type OmpT protease to release human adrenocorticotropic hormone(1-24). It was also cleaved at Arg.sup.140-Arg.sup.141 to release RAR-ACTH. Though not shown in
(115) No by-products were released by cleavage at other sites. The fusion protein PCT was cleaved at Arg.sup.139-Arg.sup.140 and Arg.sup.140-Arg.sup.141 by wild-type OmpT protease, releasing RRAR-CT (SEQ ID NO: 13) and RAR-CT. PCT was cleaved at Arg.sup.139-Arg.sup.140, Arg.sup.141-Ala.sup.142 and Arg.sup.143-Cys.sup.144 by D97H, releasing RRAR-CT (SEQ ID NO: 13), AR-CT and human calcitonin precursor. Release of the target physiologically active polypeptide by wild-type OmpT protease was confirmed from all of the fusion proteins. This demonstrated that physiologically active polypeptide production systems utilizing linker polypeptide sequences and OmpT protease variants indicated in the examples can be applied not only for specific physiologically active polypeptides, and therefore the general utility of this method is thought to be considerable.
Example 17
Co-Expression of Fusion Protein PMT and OmpT Protease Variant D97M
(116) When a fusion protein is expressed as inclusion bodies in E. coli and the host E. coli cells express OmpT protease, cleavage by OmpT protease occurs simply by dissolving the obtained inclusion bodies in urea. It was therefore investigated whether human motilin can be released by inclusion body lysis when the fusion protein PMT-expressing plasmid pG117S4HompPMT (see Example 9) and a OmpT protease variant D97M-expressing plasmid are co-expressed using OmpT protease-deficient E. coli W3110 M25 as the host cells. The OmpT protease variant D97M-expressing plasmid pOmpTD97M is incompatible because it has the same replication origin as pG117S4HompPMT.
(117) An OmpT protease variant D97M-expressing plasmid from pMW218 (
(118) The W3110 M25 recombinant E. coli was subjected to gyratory culturing at 37 C. overnight in a 2 L Erlenmeyer flask using 400 mL of LB broth containing 10 mg/L tetracycline and 20 mg/L kanamycin. The inclusion bodies were prepared according to ordinary protocol, except that all washing was performed with deionized water. The reaction for release of human motilin from the obtained inclusion bodies was carried out in the following manner. After adding 20 L of 1 M sodium phosphate (pH 7.0) and 16 L of 50 mM EDTA to 160 L of 10 M urea, 80 L of fusion protein inclusion bodies (OD.sub.660=100) was added for lysis of the inclusion bodies. Water (124 L) was then added to start the reaction.
(119) The reaction was carried out at 25 C., and sampling was performed at 20, 40, 60, 120, 180, 240, 300, 360 and 1440 minutes after start of the reaction for analysis by SDS-PAGE (
Example 18
Reaction Between Fusion Proteins PMT, PMT6D and PMT7D, and OmpT Protease Variant D97M
(120) While the results of Example 14 demonstrated that motilin is produced from the fusion protein PMT by the OmpT protease variant D97M, cleavage also occurred at Arg.sup.139-Arg.sup.140 to yield the by-product RRAR-motilin (SEQ ID NO: 13). On the other hand, the results of Example 8 indicated that situating the acidic amino acid aspartic acid at position P3 or P4 where cleavage is not desired can inhibit cleavage at those sites.
(121) Thus, since Arg.sup.139-Arg.sup.140 of the motilin fusion protein PMT is a site where cleavage is not desired, plasmids were constructed having the structures shown in
(122) Elution was performed for 40 minutes with a linear gradient of 20-60% acetonitrile containing 0.1% trifluoroacetic acid, and absorption at 220 nm was monitored. The inclusion body protein concentration was detected using bovine serum albumin (BSA) as the standard sample. The OmpT protease variant D97M suspension (OD.sub.660=0.5) in the E. coli outer membrane fraction was supplied to SDS-PAGE, and the variant concentration was measured with a densitometer using purified OmpT as the standard sample. The results of HPLC analysis of each of the reaction solutions are shown in
(123)
(124) The basic full amino acid sequences of each of the fusion proteins according to the invention are listed below.
(125) Fusion Protein PRR (SEQ ID NO: 1;
(126) TABLE-US-00004 SequenceforPRR MetThrMetIleThrAspSerLeuAlaValValLeuGlnArgLys 15 AspTrpGluAsnProGlyValThrGlnLeuAsnArgLeuAlaAla 30 HisProProPheAlaSerTrpArgAsnSerAspAspAlaArgThr 45 AspArgProSerGlnGlnLeuArgSerLeuAsnGlyGluTrpArg 60 PheAlaTrpPheProAlaProGluAlaValProGluSerLeuLeu 75 AspLeuProGluAlaAspThrValValValProAspSerSerAsn 90 TrpGlnMetHisGlyTyrAspAlaProIleTyrThrAsnValThr 105 TyrProIleThrValAsnProProPheValProThrGluProHis 120 HisHisHisProGlyGlyArgGlnMetHisGlyTyrAspAlaGlu 135 LeuArgLeuTyrArgArg HisHisGlySerGlySerProTyrArg 150 HisProArgHisAlaGluGlyThrPheThrSerAspValSerSer 165 TyrLeuGluGlyGlnAlaAlaLysGluPheIleAlaTrpLeuVal 180 LysGlyArgGly 184
(127) In this amino acid sequence, the underlined portion is the amino acid sequence of human glucacon-like peptide-1(7-37) (GLP-1(7-37)), and the double underlined portion is the basic amino acid pair (Arg.sup.140-Arg.sup.141) which is the OmpT protease cleavage site. The protecting protein (-gal117S4H) derived from the 117 N-terminal amino acids of -galactosidase of E. coli consists of the amino acid sequence from methionine at amino acid No. 1 to arginine at amino acid No. 127. The linker peptide consists of the amino acid sequence from glutamine at amino acid No. 128 to arginine at amino acid No. 153.
(128) PA-Derivative Fusion Protein (SEQ. ID No: 2;
(129) TABLE-US-00005 SequenceforPA MetThrMetIleThrAspSerLeuAlaValValLeuGlnArgLys 15 AspTrpGluAsnProGlyValThrGlnLeuAsnArgLeuAlaAla 30 HisProProPheAlaSerTrpArgAsnSerAspAspAlaArgThr 45 AspArgProSerGlnGlnLeuArgSerLeuAsnGlyGluTrpArg 60 PheAlaTrpPheProAlaProGluAlaValProGluSerLeuLeu 75 AspLeuProGluAlaAspThrValValValProAspSerSerAsn 90 TrpGlnMetHisGlyTyrAspAlaProIleTyrThrAsnValThr 105 TyrProIleThrValAsnProProPheValProThrGluProHis 120 HisHisHisProGlyGlyArgGlnMetHisAlaAlaAlaAlaAla 135 AlaAlaAlaAlaArgArg AlaAlaAlaAlaGlySerProTyrArg 150 HisProArgHisAlaGluGlyThrPheThrSerAspValSerSer 165 TyrLeuGluGlyGlnAlaAlaLysGluPheIleAlaTrpLeuVal 180 LysGlyArgGly 184
(130) In this amino acid sequence, the underlined portion is the amino acid sequence of human glucacon-like peptide-1(7-37) (GLP-1(7-37)), and the double underlined portion is the basic amino acid pair (Arg.sup.140-Arg.sup.141) which is the OmpT protease cleavage site. The protecting protein (-gal117S4H) derived from the 117 N-terminal amino acids of -galactosidase of E. coli consists of the amino acid sequence from methionine at amino acid No. 1 to arginine at amino acid No. 127. The linker peptide consists of the amino acid sequence from glutamine at amino acid No. 128 to arginine at amino acid No. 153.
(131) PA3-Derivative Fusion Protein (SEQ. ID No: 3;
(132) TABLE-US-00006 SequenceforPA3 MetThrMetIleThrAspSerLeuAlaValValLeuGlnArgLys 15 AspTrpGluAsnProGlyValThrGlnLeuAsnArgLeuAlaAla 30 HisProProPheAlaSerTrpArgAsnSerAspAspAlaArgThr 45 AspArgProSerGlnGlnLeuArgSerLeuAsnGlyGluTrpArg 60 PheAlaTrpPheProAlaProGluAlaValProGluSerLeuLeu 75 AspLeuProGluAlaAspThrValValValProAspSerSerAsn 90 TrpGlnMetHisGlyTyrAspAlaProIleTyrThrAsnValThr 105 TyrProIleThrValAsnProProPheValProThrGluProHis 120 HisHisHisProGlyGlyArgGlnMetHisAlaAlaAlaAlaAla 135 AlaAlaAlaAlaArgArg AlaArgAla AlaGlySerProTyrArg 150 HisProArgHisAlaGluGlyThrPheThrSerAspValSerSer 165 TyrLeuGluGlyGlnAlaAlaLysGluPheIleAlaTrpLeuVal 180 LysGlyArgGly 184
(133) In this amino acid sequence, the underlined portion is the amino acid sequence of human glucacon-like peptide-1(7-37) (GLP-1(7-37)), and the double underlined portions are the OmpT protease cleavage sites (Arg.sup.140-Arg.sup.141 and Arg.sup.143-Ala.sup.144). The protecting protein (-gal117S4H) derived from the 117 N-terminal amino acids of -galactosidase of E. coli consists of the amino acid sequence from methionine at amino acid No. 1 to arginine at amino acid No. 127. The linker peptide consists of the amino acid sequence from glutamine at amino acid No. 128 to arginine at amino acid No. 153.
(134) PA23-Derivative Fusion Protein (SEQ. ID No: 4;
(135) TABLE-US-00007 SequenceforPA23 MetThrMetIleThrAspSerLeuAlaValValLeuGlnArgLys 15 AspTrpGluAsnProGlyValThrGlnLeuAsnArgLeuAlaAla 30 HisProProPheAlaSerTrpArgAsnSerAspAspAlaArgThr 45 AspArgProSerGlnGlnLeuArgSerLeuAsnGlyGluTrpArg 60 PheAlaTrpPheProAlaProGluAlaValProGluSerLeuLeu 75 AspLeuProGluAlaAspThrValValValProAspSerSerAsn 90 TrpGlnMetHisGlyTyrAspAlaProIleTyrThrAsnValThr 105 TyrProIleThrValAsnProProPheValProThrGluProHis 120 HisHisHisProGlyGlyArgGlnMetHisAlaAlaAlaAlaAla 135 AlaAlaAlaArgArgArg AlaArgAla AlaGlySerProTyrArg 150 HisProArgHisAlaGluGlyThrPheThrSerAspValSerSer 165 TyrLeuGluGlyGlnAlaAlaLysGluPheIleAlaTrpLeuVal 180 LysGlyArgGly 184
(136) In this amino acid sequence, the underlined portion is the amino acid sequence of human glucacon-like peptide-1(7-37) (GLP-1(7-37)), and the double underlined portions are the OmpT protease cleavage sites (Arg.sup.139-Arg.sup.140, Arg.sup.140-Arg.sup.141 and Arg.sup.143-Ala.sup.144). The protecting protein (-gal117S4H) derived from the 117 N-terminal amino acids of -galactosidase of E. coli consists of the amino acid sequence from methionine at amino acid No. 1 to arginine at amino acid No. 127. The linker peptide consists of the amino acid sequence from glutamine at amino acid No. 128 to arginine at amino acid No. 153.
(137) Fusion Protein PRMT (SEQ. ID No: 5;
(138) TABLE-US-00008 SequenceforPRMT MetThrMetIleThrAspSerLeuAlaValValLeuGlnArgLys 15 AspTrpGluAsnProGlyValThrGlnLeuAsnArgLeuAlaAla 30 HisProProPheAlaSerTrpArgAsnSerAspAspAlaArgThr 45 AspArgProSerGlnGlnLeuArgSerLeuAsnGlyGluTrpArg 60 PheAlaTrpPheProAlaProGluAlaValProGluSerLeuLeu 75 AspLeuProGluAlaAspThrValValValProAspSerSerAsn 90 TrpGlnMetHisGlyTyrAspAlaProIleTyrThrAsnValThr 105 TyrProIleThrValAsnProProPheValProThrGluProHis 120 HisHisHisProGlyGlyArgGlnMetHisGlyTyrAspAlaGlu 135 LeuArgLeuTyrArg PheValProIlePheThrTyrGlyGlyLeu 150 GlnArgMetGlnGluLysGluArgAsnLysGlyGln 162
(139) In this amino acid sequence, the underlined portion is the amino acid sequence of human motilin, and the double underlined portion is the arginine (Arg.sup.140) corresponding to position P1 of the OmpT protease cleavage site of the fusion protein PRR. The protecting protein (-gal117S4H) derived from the 117 N-terminal amino acids of -galactosidase of E. coli consists of the amino acid sequence from methionine at amino acid No. 1 to arginine at amino acid No. 127. The linker peptide consists of the amino acid sequence from glutamine at amino acid No. 128 to arginine at amino acid No. 140.
(140) Fusion protein PMT (SEQ. ID No: 6;
(141) TABLE-US-00009 SequenceforPMT MetThrMetIleThrAspSerLeuAlaValValLeuGlnArgLys 15 AspTrpGluAsnProGlyValThrGlnLeuAsnArgLeuAlaAla 30 HisProProPheAlaSerTrpArgAsnSerAspAspAlaArgThr 45 AspArgProSerGlnGlnLeuArgSerLeuAsnGlyGluTrpArg 60 PheAlaTrpPheProAlaProGluAlaValProGluSerLeuLeu 75 AspLeuProGluAlaAspThrValValValProAspSerSerAsn 90 TrpGlnMetHisGlyTyrAspAlaProIleTyrThrAsnValThr 105 TyrProIleThrValAsnProProPheValProThrGluProHis 120 HisHisHisProGlyGlyArgGlnMetHisAlaAlaAlaAlaAla 135 AlaAlaAlaArgArgArgAlaArgPheValProIlePheThrTyr 150 GlyGluLeuGlnArgMetGlnGluLysGluArgAsnLysGlyGln 165
(142) In this amino acid sequence, the underlined portion is the amino acid sequence of human motilin. The protecting protein (-gal117S4H) derived from the 117 N-terminal amino acids of -galactosidase of E. coli consists of the amino acid sequence from methionine at amino acid No. 1 to arginine at amino acid No. 127. The linker peptide consists of the amino acid sequence from glutamine at amino acid No. 128 to arginine at amino acid No. 143.
(143) Fusion Protein PAC (SEQ. ID No: 7;
(144) TABLE-US-00010 SequenceforPAC MetThrMetIleThrAspSerLeuAlaValValLeuGlnArgLys 15 AspTrpGluAsnProGlyValThrGlnLeuAsnArgLeuAlaAla 30 HisProProPheAlaSerTrpArgAsnSerAspAspAlaArgThr 45 AspArgProSerGlnGlnLeuArgSerLeuAsnGlyGluTrpArg 60 PheAlaTrpPheProAlaProGluAlaValProGluSerLeuLeu 75 AspLeuProGluAlaAspThrValValValProAspSerSerAsn 90 TrpGlnMetHisGlyTyrAspAlaProIleTyrThrAsnValThr 105 TyrProIleThrValAsnProProPheValProThrGluProHis 120 HisHisHisProGlyGlyArgGlnMetHisAlaAlaAlaAlaAla 135 AlaAlaAlaArgArgArgAlaArgSerTyrSerMetGluHisPhe 150 ArgTrpGlyLysProValGlyLysLysArgArgProValLysVal 165 TyrPro 167
(145) In this amino acid sequence, the underlined portion is the amino acid sequence of human adrenocorticotropic hormone(1-24). The protecting protein (-gal117S4H) derived from the 117 N-terminal amino acids of -galactosidase of E. coli consists of the amino acid sequence from methionine at amino acid No. 1 to arginine at amino acid No. 127. The linker peptide consists of the amino acid sequence from glutamine at amino acid No. 128 to arginine at amino acid No. 143.
(146) Fusion Protein PCT (SEQ. ID No: 8;
(147) TABLE-US-00011 SequenceforPCT MetThrMetIleThrAspSerLeuAlaValValLeuGlnArgLys 15 AspTrpGluAsnProGlyValThrGlnLeuAsnArgLeuAlaAla 30 HisProProPheAlaSerTrpArgAsnSerAspAspAlaArgThr 45 AspArgProSerGlnGlnLeuArgSerLeuAsnGlyGluTrpArg 60 PheAlaTrpPheProAlaProGluAlaValProGluSerLeuLeu 75 AspLeuProGluAlaAspThrValValValProAspSerSerAsn 90 TrpGlnMetHisGlyTyrAspAlaProIleTyrThrAsnValThr 105 TyrProIleThrValAsnProProPheValProThrGluProHis 120 HisHisHisProGlyGlyArgGlnMetHisAlaAlaAlaAlaAla 135 AlaAlaAlaArgArgArgAlaArgCysGlyAsnLeuSerThrCys 150 MetLeuGlyThrTyrThrGlnAspPheAsnLysPheHisThrPhe 165 ProGlnThrAlaIleGlyValGlyAlaProGly 176
(148) In this amino acid sequence, the underlined portion is the amino acid sequence of human calcitonin precursor. The protecting protein (-gal117S4H) derived from the 117 N-terminal amino acids of -galactosidase of E. coli consists of the amino acid sequence from methionine at amino acid No. 1 to arginine at amino acid No. 127. The linker peptide consists of the amino acid sequence from glutamine at amino acid No. 128 to arginine at amino acid No. 143.