Stable troponin subunits and complexes

FIELD OF THE INVENTION
This invention relates to recombinantly-expressed human cardiac troponin subunits I, C, and T, recombinant human cardiac troponin complexes made in vitro therefrom, and methods for the production and purification of the subunits and the preparation of the complexes.
BACKGROUND OF THE INVENTION
Early and accurate assessment of suspected acute myocardial infarction is critically dependent on the sensitive and specific detection and quantitation in blood, serum or plasma of released cardiac muscle intracellular components in order to distinguish a potentially lethal event in need of emergency measures from non-life threatening conditions such as angina and non-cardiac chest pain such as dyspepsia. Early electrocardiographic changes are not adequately specific, and the medical profession has come to rely on serum biochemical markers of cardiac tissue necrosis for early diagnosis. Initially, the serum markers creatine kinase (CK) and specifically the cardiac CK-MB isoform were used, and subsequently myoglobin as a more sensitive early indicator of cardiac damage. More recently, the highly sensitive markers cardiac troponin subunits I and T have come to be preferred for their extraordinarily high specificity for identifying myocardial damage. These tests, along with other markers of skeletal muscle necrosis, provide a high degree of diagnostic accuracy. If performed in the emergency room an early and accurate diagnosis of myocardial damage offers great advantage to a suspected heart attack victim.
Diagnostic tests employing highly sensitive cardiac markers are described, for example, in U.S. Pat. Nos. 5,604,105 and 5,290,678. These procedures offer the rapidity of diagnosing myocardial infarction in the emergency room setting and offer significant medical benefit for patients.
Though numerous diagnostic assays for troponin subunits, principally I and T, exist, for example the troponin I Stratus(R) test from Dade International, Inc., the Opus(R) test from Behring, and the Access(R) test from Sanofi, there is no troponin subunit or complex preparation of sufficient quality and stability to employ as a standard for calibration of assays nor to use as a universally acceptable consensus standard across the industry. In order to maintain the conformational structure of troponin I, it must be complexed with troponin C. Troponin preparations from one source and for one particular assay format may not be useable in another, thus it is not currently possible to use the same standard across tests from different manufacturers. The lack of a industry-accepted standard limits the establishment of industry guidelines for test criteria as well as comparison of results throughout the world to help establish normal and abnormal value ranges.
Numerous troponin preparations from both natural and recombinant sources have been proposed. Previously described methods for the purification of troponin subunits from cardiac tissue (Tsukui et al., 1973, J. Biochem., v. 73, pp. 1119-1121; Cummins et al., 1978, Biochem. J., v. 171, pp. 251-259; Syska et al., 1974, FEBS Letts., v. 40, pp. 253-257) yielded preparations which were unstable and subject to considerable degradation on storage. A troponin complex prepared from cardiac tissue or from the combination of isolated troponin subunits prepared from cardiac tissue has been described (EP 0 743 522 A1). DE4405249 (U.S. Pat. No. 5,583,200) describes a stabilized troponin I or T also containing troponin C, with stability of several days in the cold. Use of a combination of four protease inhibitors in U.S. Pat. No. 5,560,937 overcame the degradation and stability issues inherent in tissue-derived troponin I, but did not obviate the need for human heart tissue. Unfortunately, human tissue is a potential source of infection, including HIV and hepatitis, to workers during troponin purification, and human heart tissue is of increasing scarcity especially with the successful use of early diagnostic tests for heart attack responsible for decreasing mortality from this disease. Troponin standards prepared by recombinant means offer a less costly and less hazardous alternative to that from heart tissue, but may still suffer from a lack of stability incompatible with industry demands.
Recombinant troponin subunits and complexes have been described. Armour et al. (1993, Gene, v. 131. pp. 287-292) cloned human cardiac troponin I in a bacterial system and expressed the gene product both as a beta-gal fusion product, and as an unfused product. Al-Hillawi et al. (1994, Eur. J. Biochem., v. 225, pp. 1195-1201) expressed human cardiac troponin I and troponin C in E. coli, using two codon changes in the cDNA of the former to overcome difficulties in expressing the human product in bacteria. Malnic and Reinach (1994, Eur. J. Biochem., v. 222, pp. A9-54) produced a recombinant complex in vivo by cloning all three chicken skeletal muscle troponin subunits into an expression plasmid. The troponin complex formed within the bacterium. Fujita-Becker et al. (1993, J. Biochem., v. 114, pp. 438-444) described the reconstitution of rabbit skeletal troponin complex from recombinant subunits expressed in E. coli. None of these recombinant products has been demonstrated to have adequate stability for use as a diagnostic test standard or calibrator. A recombinant human cardiac troponin complex formed in vitro from recombinant human cardiac troponin I, recombinant human cardiac troponin T, and recombinant human cardiac troponin C has not been described previously, nor has a complex formed of recombinant human cardiac troponin I and recombinant human cardiac troponin C.
Thus, there is a need for troponin complexes which meet the requirements of a stable material of safe origin and economical preparation that may be used as controls or calibrators across troponin assays. It has now been discovered that human cardiac troponin complexes prepared from recombinant subunits offer, among other advantages, superior stability and utility among troponin assays to be suitable for use as a universal standard.
SUMMARY OF THE INVENTION
The present invention relates to a recombinant human cardiac troponin complex comprising a recombinant modified human cardiac troponin I, recombinant human cardiac troponin C, and recombinant human cardiac troponin T, the complex formed in vitro from the individual recombinant troponin subunits. The invention also comprises a complex formed of recombinant modified human cardiac troponin I and recombinant human cardiac troponin C. The present invention also relates to a novel recombinant human cardiac troponin I subunit with a modified cDNA and protein sequence with improved expression in E. coli. The troponin complexes prepared in vitro from the recombinantly expressed subunits are of higher stability than previously reported troponin complexes prepared from recombinant methods or from troponin subunits isolated from cardiac tissue. They can be used as controls or calibrators among different troponin assay procedures. In contrast, previously known troponins cannot be so employed.
The subunits and complexes are also useful as antigens to prepare antibodies useful for a variety of purposes.
The troponin subunits are prepared by recombinant techniques, expressed and optionally purified individually before admixture to prepare the complexes. The complex may be purified by standard procedures after they are formed. Recombinant expression of the troponin subunits can be optimized. Troponin I is engineered as a recombinant product with an additional N-terminal portion of from about 4 to about 12, preferably 5 to 8, amino acids. It has been observed that the additional amino acids increase the expression of modified troponin I in E. coli. Troponin T has been expressed at high levels by altering arginine codons in the cDNA to be compatible with the bacterial expression system, without altering the amino acid sequence of the subunit. Recombinant troponin C is expressed with the native amino acid sequence.
Modified Troponin I as used herein refers to Troponin I with additional N-terminal acids to increase the expression of the desired product. It is not essential that the amino acids be identical with or in the same order as shown in the figure.
The subunits are prepared separately before being brought together in vitro under selected conditions to form the troponin complexes. Conditions are selected to promote complex formation, including the use of an alkaline earth salt or salts and, if desired, a detergent for the complex of troponins C, I and T. One or more alkaline earth salts, and a chaotropic agent, may be employed in the preparation of the complex of troponin C and I. Calcium chloride and magnesium chloride are preferred as alkaline earth salts. As the detergent, sodium dodecyl sulfate is preferred. Urea is the preferred chaotropic agent.
It is an objective of the present invention to provide a recombinant human cardiac troponin I protein of SEQ ID NO: 5 which has an additional N-terminus of about 6 amino acids and other codon changes which facilitates the recombinant expression of the protein. It is a further objective of this invention to provide a recombinant human cardiac troponin T protein produced from a cDNA sequence set forth in SEQ ID NO: 4 which has certain codon changes in the cDNA but no change in protein sequence compared to native human cardiac troponin T.
According to the present invention, a method is provided for the preparation of human cardiac troponin complexes prepared from recombinant modified troponin I, recombinant troponin C, and recombinant troponin T. The method comprises the steps of: 1) expressing the troponin subunits recombinantly; 2) optionally purifying the separate subunits; 3) admixing the subunits preferably in the presence of at least one alkaline earth salt or salts and, if desired, a detergent, to form the troponin complex. The use of a detergent increases the yield and purity of the products. It is a further object of the invention to provide a method for the preparation of a human cardiac troponin complex prepared from recombinant modified troponin I and recombinant troponin C, comprising the steps of: 1) expressing the troponin subunits recombinantly; 2) admixing the subunits preferably in the presence of an alkaline earth salt or salts and a chaotropic agent to form the troponin complex. Purification is preferably effected before formation of a complex. The present invention contemplates modifications to the DNA sequence of the troponin subunits to facilitate their expression in their respective recombinant systems.
The recombinant troponin complexes according to the present invention have the purity and stability characteristics to enable utility as assay controls and calibrators for use in the diagnostics industry for the manufacture, quality control, and calibration of troponin assays. The troponin complexes of the present invention show superior stability compared to existing troponin complexes, and may be utilized as controls among different troponin assay procedures, in contrast to existing troponin controls which cannot be used except in the specific assays for which they were designed.
It is a further advantage of the present invention that production of the recombinant human cardiac troponin complexes is independent of the availability of human cardiac tissue. Working with human tissues presents an inherent risk of infection by any number of diseases including HIV and hepatitis. In addition, the quality of tissue may be unknown resulting in the production of an inferior product. Heart tissue from elderly individuals may be hypertrophic and as a result have a low muscle mass and thus not provide a satisfactory quantity or quality of troponin. Human heart tissue is prohibitively costly as a long-term, consistent source of troponin to meet the future demands of the rapidly expanding field of emergency cardiac diagnostics.
Another advantage of the present invention is the ease of purification compared to products isolated from human cardiac tissue. Consequently, the authenticity and purity of each subunit can be simply determined prior to the preparation of the complexes. The lack of uniformity of tissue, presence of degradation or autolysis products, etc., are obviated by use of the individual recombinant subunits rather than products isolated from natural sources.
A further advantage of the present invention is that it provides a novel recombinant modified human cardiac troponin I that may be used as an assay control of calibrator.
A still further advantage is that the invention provides recombinant troponin subunits of sufficient purity and quantity to permit their use as controls or calibrators and also to serve as substrates from which to produce stable troponin complexes that meet the purity and stability requirements to be used as an assay standard or calibrator.
These and other advantages of the recombinant human cardiac troponin complexes and its subunits in the present invention will be apparent upon consideration of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1B represent the DNA (SEQ ID NO: 1) and protein (SEQ ID NO: 5) sequences of the recombinant modified human cardiac troponin I of the present invention. The box FIG. 1A points out the six amino acid residues added to the N-terminus of human cardiac troponin I. The original bases (SEQ ID NO: 3, including the additional N-terminal amino acids) are shown above the altered sequences.
FIGS. 2A-2C represent the DNA (SEQ ID NO: 4) and protein (SEQ ID NO: 6) sequences of the recombinant human cardiac troponin T of the present invention. The codons which replace the natural codon is underlined FIG. 2B, and the natural codon is shown above. The native troponin T DNA sequence is shown in SEQ ID NO: 2.

DETAILED DESCRIPTION OF THE INVENTION
Measurement in circulation of the cardiac muscle-associated protein troponin and, particularly, its components I and T, has proven to be an early and specific indicator of suspected acute myocardial infarction. As such, numerous methods for rapidly and accurately detecting troponin and its subunits in blood have been and are being developed for diagnosing heart attack in an emergency situation, and countless lives have been and will be saved as a result. However, in order to develop accurate and dependable diagnostic assays and to calibrate them, the availability of stable, high-quality human cardiac troponin controls is critical for quality control and testing purposes. This invention provides pure, stable, recombinant human cardiac troponin complexes to meet the needs of the industry. A troponin complex may be formed in vitro from the admixture of recombinantly expressed human cardiac troponin I, troponin T and troponin C. Another troponin complex may be formed from the admixture of recombinantly expressed human cardiac troponin I and C.
Troponin I may be engineered as a recombinant product with about 6 additional amino acid N-terminal residues (see FIGS. 1A-1B; SEQ ID NO: 5) to increase its expression in E. coli. In addition, certain codon changes have been made in the cDNA sequence to optimize expression of the protein in E. coli, without change in the amino acid sequence. The modified recombinant troponin I may be isolated from the bacterial host and purified by standard protein purification methods following details illustrated in the examples below. The purified modified troponin I is then be employed as an antigen or to prepare the recombinant troponin complexes such as that of troponin I, troponin C and troponin T, and that of troponin I and troponin C. Troponin C stabilizes the conformational structure of troponin I. In addition, the recombinant, modified human cardiac troponin I has utility itself as an improved control or calibrator. This E. coli strain was deposited on Oct. 30, 1997, with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, and accorded ATCC number 98567.
Recombinant troponin T has been expressed at high levels by altering arginine codons in the cDNA (see FIGS. 2A-2C; SEQ ID NO:4) to be compatible with the bacterial expression system, without altering the amino acid sequence of the subunits. The recombinant troponin T is isolated from the bacterial host and, optionally, purified by standard protein purification methods following details in the example below. The recombinant troponin T produced by this method has utility itself as an improved control or calibrator. The E. coli strain was deposited on Oct. 30, 1997 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, and accorded ATCC number 98566.
The troponin C is expressed in E. coli in its native amino acid sequence, then isolated from the bacterial host and optionally purified by standard protein purification methods following details illustrated in the examples below.
Typically, the subunits are prepared separately and optimally purified before being brought together in vitro under selected conditions to form the complex of troponin I, T and C. Conditions are selected to promote complex formation, including the use of an alkaline earth salt or salts and, if desired, a detergent. As the alkaline earth salts, calcium chloride and magnesium chloride are preferred. As the detergent, sodium dodecyl sulfate is preferred. For the complex of troponin I and C, conditions are selected to promote complex formation including the use of an alkaline earth salt or salts and a chaotropic agent. As the alkaline earth salts, calcium chloride and magnesium chloride are preferred. As the chaotropic agent, urea is preferred.
As will be evident from the examples presented below, the recombinant troponin complexes according to the present invention have purity and stability characteristics to enable their utility as assay controls and calibrators for use in the diagnostics industry for the manufacture, quality control, and calibration of troponin assays. The troponin complexes of the present invention shows superior stability compared to known troponin complexes, and are shown to be detected utilizing different troponin assay methods and instruments, in contrast to existing troponin controls which cannot be used as a standard outside of the specific assay for which they were designed.
The preparation of the recombinant troponin subunits, their purification, and preparation of the complexes are illustrated in the following non-limiting examples, which also demonstrate their stability and utility in various assays.
EXAMPLE 1
Expression and Purification of Modified Human Cardiac Troponin I
Human cardiac troponin I cDNA was cloned by the polymerase chain reaction (PCR) using primers designed from published cardiac troponin I cDNA sequence (Vallins et al., 1990, FEBS Letters, vol. 270, pp. 57-61, the full disclosure of which is incorporated herein by reference). Since the expression of human cardiac troponin I was very low in E. coli if the original cDNA sequence was used (Armour et al., 1993, Gene, vol. 131, pp. 287-292), both N-terminus addition and codon alteration were performed to increase the yield. The altered human cardiac troponin I cDNA sequence (FIG. 1; SEQ ID NO: 1) was confirmed by DNA sequencing and cloned into plasmid pET21 (Novagen). E. coli BL21 (DE3) cells (Novagen) were transformed with the resulting construct and the protein produced from this expression vehicle analyzed using SDS-PAGE. This E. coli strain has been deposited on Oct. 30, 1997, with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, and accorded ATCC number 96567.
For purification, the following reagents were used: lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 20 mM 2-mercaptoethanol, 100 mM sodium chloride, pH 8.0); 2 M urea-lysis buffer (2 M urea in lysis buffer; 8 M urea-lysis buffer (8 M urea in lysis buffer); dialysis buffer (20 mM Tris-HCl, 100 mM sodium chloride, 10 mM 2-mercaptoethanol. pH 7.5); eluting buffer (20 mM Tris-HCl, 800 mM sodium chloride, 10 mM 2-mercaptoethanol. pH 7.5): final buffer (20 mM Tris-HCl, 500 mM sodium chloride, 60 mM 2-mercaptoethanol, pH 7.5).
The following sequential steps were carried out for the purification of the modified human recombinant troponin I protein, Recombinant bacterial cells were collected by centrifugation of the culture medium. Cells were resuspended in lysis buffer at the ratio of 50 ml lysis buffer per liter equivalent of original culture medium. Lysozyme (10 mg/ml) was added at 1 ml lysozyme per liter of culture medium. The resulting mixture was then shaken at room temperature for 30 minutes. Triton X-100 (10%) was added at 5 ml per liter of culture medium, followed by PMSF (0.1 M) at 0.5 ml per liter of medium. The suspension was incubated at 37 C. for 15 minutes. The vessel containing the suspension was submerged in dry ice/ethanol to rapidly freeze the cell suspension. The frozen suspension was then thawed in a 37 C. water bath. The thawed cell suspension was sonicated using a Vibra Cell.TM. sonicator at 2 C.-8 C. to shear the DNA. The suspension was centrifuged at 20,000 g for 20 min at 2 C.-8 C. and the supernatant was discarded. Fifty ml of 2 M urea-lysis buffer and 10 ml of 10% Triton X-100 was added per liter equivalent of original culture medium; the pellet was homogenized and stirred at 4 C. for 15 min. The mixture was then centrifuged at 20,000 g for 20 min at 2 C.-8 C. and the supernatant discarded. 8 M urea-lysis buffer (50 ml of urea-lysis buffer per liter cell culture) was added, and the mixture homogenized then stirred for 30 min at room temperature. After centrifugation at 20,000 g at 2 C.-8 C. for 20 min. the pellet is discarded. The supernatant is dialyzed in dialysis buffer overnight, and if required, the dialysate was centrifuged to remove any particulate matter.
The sample was loaded onto a previously equilibrated CM-Sephadex column (for example, a 2.5.times.20 cm; 100 ml bed volume for 2 L of original culture medium). The column was washed with 4 bed volumes of the dialysis buffer, and eluted with a linear gradient of 3.5 bed volumes of 0-800 mM NaCl (100% dialysis buffer changed over to 100% eluting buffer). The fractions were checked by 15% SDS-PAGE and those containing the troponin I were pooled then dialyzed against final buffer. The dialysate was centrifuged at 20,000 g for 20 min and the supernatant filtered through two layers of Miracloth. In order to remove endotoxin from the protein preparation and form a product useful as an antigen, following Triton X-114 phase separation procedures are performed. Triton X-114 (100%) was added at 1 ml per 100 ml of the supernatant and stirred at 4 C. for 30 min. The sample was incubated at 37 C. for 10 min., then centrifuged at 20,000 g for 5 min at 30 C. using 40-ml centrifuge tubes. After removing the upper aqueous phase with care so as not to aspirate the detergent phase, the aqueous phases were combined. The addition and centrifugation steps with Triton X-114 were repeated two more times. The aqueous phase was dialyzed overnight against the final buffer (100 ml sample per liter of the final buffer).
Final determination of protein concentration was performed using the Bradford Assay, using Bradford BSA as the standard, and final purity using a 15% SDS-PAGE. The purified recombinant human cardiac troponin I was stored at -20 C.
EXAMPLE 2
Expression and purification of troponin T
Human cardiac troponin T was cloned and expressed as previously described (Hu et al., 1996, Protein Expression and Purification, vol. 7, pp. 289-293, the full disclosure of which is incorporated herein by reference). According to this method, the codons of two consecutive pairs of AGG codons in the human cardiac troponin T cDNA were replaced in order to achieve a high level of expression in E. coli. The replacements were with synonymous codons thus the protein produced from this expression vehicle was not altered (see FIG. 2; SEQ ID NO: 6). A 40-fold increased expression was achieved. This E. coli strain has been deposited on Oct. 30, 1997, with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, and accorded ATTC number 98566.
For purification of recombinant troponin T, the following reagents were used: lysis buffer: (50 mM Tris-HCl, 1 mM EDTA, 20 mM 2-mercaptoethanol, 100 mM sodium chloride, pH 8.0); dialysis buffer 1 (50 mM Tris-HCl, 500 mM potassium chloride, 5 mM 2-mercaptoethanol, 1 mM EDTA, pH 8.0); dialysis buffer 11 (50 mM Tris-HCl, 5 mM 2-mercaptoethanol, pH 8.0); eluting buffer (50 mM Tris-HCl, 500 mM sodium chloride, 5 mM 2-mercaptoethanol, pH 8); storage buffer (50 mM Tris-HCl, 100 mM sodium chloride, 5 mM 2-mercaptoethanol, pH 7.5).
Purification of recombinant troponin T was accomplished as follows. Recombinant bacterial cells were collected by centrifugation of the cell culture medium. Cells were resuspend in lysis buffer at the ratio of 50 ml lysis buffer per liter equivalent of culture medium. Lysozyme (10 mg/ml) was added at 1 ml lysozyme per liter of culture medium. For antigen preparation, the resulting mixture was then shaken at room temperature for 30 minutes. Triton X-100 (10%) was added at 5 ml per liter of original culture medium, followed by PMSF (0.1 M) at 0.5 ml of PMSF per liter of medium. The suspension was incubated at 37 C. for 15 minutes. The vessel containing the suspension was submerged into dry ice/ethanol to rapidly freeze the cell suspension. The frozen suspension was then thawed in a 37 C. water bath. The thawed cell suspension was sonicated using a Vibra Cell.TM. sonicator at 2 C.-8 C. to shear the DNA. The suspension was centrifuged at 20,000 g for 20 min at 2 C.-8 C. and the pellet was discarded. The supernatant was dialyzed in dialysis buffer I overnight, and if required, the dialysate was centrifuged to remove any particular matter.
The troponin T in the supernatant was precipitated with 30-50% ammonium sulfate by adding the required amount of solid ammonium sulfate slowly and stirring for 30 min. The precipitated protein was dissolved in and dialyzed against dialysis buffer II overnight, and then if required, centrifuged to remove any particulate matter. The sample was loaded onto a previously equilibrated DEAE-Bio-gel column (for example, 2.5.times.20 cm; 100 ml bed volume for 2 L of equivalent cell culture medium volume). After washing the column with 4 bed volumes of the dialysis buffer II, protein was eluted with a linear gradient of 3.5 bed volumes of 0-800 mM NaCl (dialysis buffer II changed over to eluting buffer). The fractions were checked using 15% SDS-PAGE and the recombinant human cardiac troponin T fractions pooled then dialyzed against storage buffer. The dialysate was then centrifuged at 20,000 g for 20 min and the supernatant filtered through two layers of Miracloth.
In order to remove endotoxin from the protein preparation to produce an antigen, following Triton X-114 phase separation procedures are performed. Triton X-114 (100%) was added at 1 ml per 100 ml of the supernatant and stirred at 4 C. for 30 min. The sample was incubated at 37 C. for 10 min., then centrifuged at 20,000 g for 5 min at 30 C. using 40-ml centrifuge tubes. After removing the upper aqueous phase with care so as not to aspirate the detergent phase, the aqueous phases were combined. The addition and centrifugation steps with Triton X-114 were repeated two more times. The aqueous phase was dialyzed overnight against the storage buffer (100 ml sample per liter of the final buffer).
Final determination of protein concentration was performed using the Bradford Assay, with BSA as the standard, and final purity using a 15% SDS-PAGE. Recombinant human cardiac troponin T was stored at -20 C.
EXAMPLE 3
Expression and Purification of Troponin C
Human cardiac troponin C cDNA was cloned using the polymerase chain reaction (PCR) using primers designed from the published cardiac troponin C cDNA sequence (GenBank AC: X07897). The cDNA sequence was confirmed by DNA sequencing and cloned into plasmid pET21(Novagen). E. coli BL21(DE3) cells (Novagen) were transformed with the resulting construct and protein expression analyzed by SDS-PAGE.
For purification, reagents included lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 20 mM 2-mercaptoethanol, 100 mM sodium chloride, pH 8.0); and storage buffer (50 mM Tris-HCl, 200 mM sodium chloride, 1 mM CaCl.sub.2, pH 7.5).
Purification of recombinant human cardiac troponin C was accomplished as follows. Recombinant bacterial cells were collected by centrifugation of the cell culture. Cells were resuspend in lysis buffer at the ratio of 50 ml lysis buffer per liter equivalent of cell culture medium. Lysozyme (10 mg/ml) was added at 1 ml per liter of culture medium. The resulting mixture was then shaken at room temperature for 30 minutes. Triton X-100 (10%) was added at 5 ml per liter of original medium, followed by PMSF (0.1 M) at 0.5 ml per liter of cell culture. The suspension was incubated at 37 C. for 15 minutes. The vessel containing the suspension was submerged into dry ice/ethanol to rapidly freeze the cell suspension. The frozen suspension was then thawed in a 37 C. water bath. The thawed cell suspension was sonicated using a Vibra Cell.TM. sonicator at 2 C.-8 C. to shear the DNA. The suspension was centrifuged at 20,000 g for 20 min at 2 C.-8 C. and the pellet was discarded. The supernatant was dialyzed in storage buffer overnight, and if required, the dialysate was centrifuged to remove any particular matter.
The supernatant was treated with 70% ammonium sulfate by adding the required amount of solid ammonium sulfate slowly and stirring for 30 min. After centrifugation, the troponin C in the supernatant was dialyzed against storage buffer overnight, then centrifuged if necessary to remove any particulate matter. The sample was concentrated in an Amicon filter to 10 ml per liter equivalent cell culture column, then was loaded on pre-equilibrated Bio-gel P60 size exclusion column. The protein was eluted with storage buffer. Fractions were checked by 15% SDS-PAGE, and the fractions containing recombinant human cardiac troponin C pooled.
In order to remove endotoxin from the protein preparation, following Triton X-114 phase separation procedures are performed. Triton X-114 (100%) was added at 1 ml per 100 ml of eluate, and the mixture was stirred at 4 C. for 30 min. Then incubated at 37 C. for 10 min. The mixture was them centrifuged at 20,000 g for 5 min. Using 40-ml centrifuge tubes, and the upper phase aspirated carefully to avoid taking the detergent phase. Aqueous phases were combined, and the detergent step repeated twice more. The aqueous phase was dialyzed against dialysis buffer overnight.
Protein concentration was determined using the Bradford Assay, with BSA as the standard. The recombinant human cardiac troponin C was lyophilized and stored at -20 C.
EXAMPLE 4
Post-purification Folding of Three Subunits Into a Troponin C-I-T Complex
The following reagents were used: 1.0 mg/ml recombinant human cardiac troponin I, 1.0 mg/ml recombinant human cardiac troponin C, and 1.0 mg/ml recombinant human cardiac troponin T, purified as described in the above examples; 2% sodium dodecyl sulfate; 400 mM CaCl.sub.2 ; 400 mM MgCl.sub.2 ; final buffer (20 mM Tris-HCl, 500 mM NaCl, 60 mM 2-mercaptoethanol, pH 7.5); storage buffer (10 mM Phosphate buffered saline, pH 6.8, 3% normal bovine serum, 4 mM CaCl.sub.2, 4 mM MgCl.sub.2, protease inhibitors, and 0.05% thimerosal).
The following procedure was employed. Ten .mu.l of 1.0 mg/ml recombinant troponin I and 10 .mu.l of 2% SDS are combined in a microcentrifuge tube. The microcentrifuge tube was placed in a boiling water bath for 5 min., then cooled at room temperature for 5 min. Ten .mu.l of 1.0 mg/ml recombinant troponin C, 10 .mu.l of 1.0 mg/ml recombinant troponin T, 10 .mu.l of 400 mM CaCl.sub.2, and 10 .mu.l of 400 mM MgCl.sub.2 were added. The mixture was shaken at room temperature for 5 min., then 940 .mu.l of the final buffer was added and the mixture shaken for an additional 25 minutes at room temperature. The above mixture was diluted into the storage buffer at 0.5 ml per 100 ml. The complex was stored at 4 C. or lyophilized.
This protocol can be scaled up proportionally to prepare larger batches of recombinant troponin complex.
EXAMPLE 5
Comparison of Recombinant Troponin Complex Stability to that of the Tissue-derived Complex
The stability of the recombinant cardiac troponin complex produced by the above procedures was compared directly to that of a human cardiac troponin complex prepared from human heart tissue in accordance with the protocol described in EP 0 743 522. The lyophilized powder form of the recombinant troponin complex was rehydrated and the stability monitored after reconstitution. Day 0 corresponds to the day of rehydration. After rehydration, the samples were stored at 4 C. and aliquots taken for assay at the indicated times. The initial (time 0) concentrations of each preparation are arbitrary and are not meant for comparison across experiments, but only with regard to stability over time. The data, expressed in ng/ml as measured by the Stratus(R) troponin I assay, are a follows:
TABLE 1______________________________________ Time Duration Storage (days)Calibrators Conditions 0 2 7 14 28______________________________________Recombinant 4 C 14.9 15.12 16.0 16.5 15.0 human cardiac troponin complex in accordance with the present invention lyophilized 10.7 N/a 10.3 11.1 10.4 powder after reconstitution and stored at 4 C Cardiac tissue 4 C 24.4 23.4 21.5 22.6 20.0 troponin complex prepared according to EP 0 743 522 lyophilized 22.4 N/a 19.4 19.5 18.5 powder after reconstitution and stored at 4 C______________________________________
As is apparent from the data presented in Table 1, the recombinant troponin complex prepared by the method of the present invention maintained its detectability by Stratus(R) assay for 28 days at 4 C., whereas the tissue-derived human cardiac troponin complex suffered a 17-18% loss of detectability during 28 days of storage.
EXAMPLE 6
Comparison of Troponin Concentration Determinations Among Various Troponin I Assays
Two commercial troponin I calibrators as well as the recombinant cardiac troponin complex prepared as described above were evaluated with two commercial troponin I assays and by ELISA. The concentration of recombinant human cardiac troponin I was based on protein mass as measured by Bradford using BSA as the standard. The results are presented in the following table.
TABLE 2______________________________________ Stated concentration Assay results (ng/ml)Troponin source (ng/ml) ELISA Stratus(R) Access(R)______________________________________Procedure 25.00 26.98 21.65 22.45 described in the present invention Stratus(R) 27.2 0 27.9 0 troponin I calibrator Access(R) 23.34 n/a 39.5 27.4 troponin I calibrator______________________________________
As is apparent from the data presented in Table 2, each troponin source was detected near its stated concentration by the assay manufactured by that troponin source; however, in both cases the commercially-available troponin I failed to assay consistently across all assays. The troponin complex of the present invention was the only preparation measurable within reasonable limits across the assays.
Similar studies with the individual subunits of the novel complex show their improved stability and utility as standards or calibrators.
EXAMPLE 7
Preparation of a Complex of Troponin I and Troponin C
The following reagents were used: 1.0 mg/ml recombinant human cardiac troponin I and 1.0 mg/ml recombinant human cardiac troponin C prepared and purified as described in the above examples, 10 M urea in distilled water, 400 mM CaCl.sub.2, and 400 mM MgCl.sub.2. The Final Buffer contained 200 mM Tris-HCl, 500 mM NaCl, and 60 mM .beta.-mercaptoethanol, pH 7.5.
The following procedure was employed. Ten .mu.l of 1.0 mg/ml troponin I and 20 .mu.l of 10 M urea were combined in a microcentrifuge tube. The tube was shaken gently for 5 minutes at room temperature. Ten .mu.l of 1.0 mg/ml troponin C, 10 .mu.l of 400 mM CaCl.sub.2, and 10 .mu.l of 400 mM MgCl.sub.2 were then added to the tube, which was then shaken gently for 5 minutes at room temperature, after which 940 .mu.l of Final Buffer was added and the tube was gently shaken at room temperature for 25 minutes. This mixture can be stored at -20 C. or lyophilized.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 6 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 651 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - ATGGCTAGCA TGGGATCTAT GGCAGACGGT TCCAGCGATG CGGCTAGGGA AC -#CTCGCCCT 60 - - GCACCAGCCC CAATCAGACG CCGCTCCTCC AACTACCGCG CTTATGCCAC GG -#AGCCGCAC 120 - - GCCAAGAAAA AATCTAAGAT CTCCGCCTCG AGAAAATTGC AGCTGAAGAC TC -#TGCTGCTG 180 - - CAGATTGCAA AGCAAGAGCT GGAGCGAGAG GCGGAGGAGC GGCGCGGAGA GA -#AGGGGCGC 240 - - GCTCTGAGCA CCCGCTGCCA GCCGCTGGAG TTGGCCGGGC TGGGCTTCGC GG -#AGCTGCAG 300 - - GACTTGTGCC GACAGCTCCA CGCCCGTGTG GACAAGGTGG ATGAAGAGAG AT -#ACGACATA 360 - - GAGGCAAAAG TCACCAAGAA CATCACGGAG ATTGCAGATC TGACTCAGAA GA -#TCTTTGAC 420 - - CTTCGAGGCA AGTTTAAGCG GCCCACCCTG CGGAGAGTGA GGATCTCTGC AG -#ATGCCATG 480 - - ATGCAGGCGC TGCTGGGGGC CCGGGCTAAG GAGTCCCTGG ACCTGCGGGC CC -#ACCTCAAG 540 - - CAGGTGAAGA AGGAGGACAC CGAGAAGGAA AACCGGGAGG TGGGAGACTG GC -#GCAAGAAC 600 - - ATCGATGCAC TGAGTGGAAT GGAGGGCCGC AAGAAAAAGT TTGAGAGCTG A - # 651 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 867 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - ATGTCTGACA TAGAAGAGGT GGTGGAAGAG TACGAGGAGG AGGAGCAGGA AG -#AAGCAGCT 60 - - GTTGAAGAGC AGGAGGAGGC AGCGGAAGAG GATGCTGAAG CAGAGGCTGA GA -#CCGAGGAG 120 - - ACCAGGGCAG AAGAAGATGA AGAAGAAGAG GAAGCAAAGG AGGCTGAAGA TG -#GCCCAATG 180 - - GAGGAGTCCA AACCAAAGCC CAGGTCGTTC ATGCCCAACT TGGTGCCTCC CA -#AGATCCCC 240 - - GATGGAGAGA GAGTGGACTT TGATGACATC CACCGGAAGC GCATGGAGAA GG -#ACCTGAAT 300 - - GAGTTGCAGG CGCTGATTGA GGCTCACTTT GAGAACAGGA AGAAAGAGGA GG -#AGGAGCTC 360 - - GTTTCTCTCA AAGACAGGAT CGAGAGACGT CGGGCAGAGC GGGCCGAGCA GC -#AGCGCATC 420 - - CGGAATGAGC GGGAGAAGGA GCGGCAGAAC CGCCTGGCTG AAGAGAGGGC TC -#GACGAGAG 480 - - GAGGAGGAGA ACCGTCGTAA GGCTGAGGAT GAGGCCCGGA AGAAGAAGGC TT -#TGTCCAAC 540 - - ATGATGCATT TTGGGGGTTA CATCCAGAAG CAGGCCCAGA CAGAGCGGAA AA -#GTGGGAAG 600 - - AGGCAGACTG AGCGGGAAAA GAAGAAGAAG ATTCTGGCTG AGCGTCGTAA GG -#TGCTGGCC 660 - - ATTGACCACC TGAATGAAGA TCAGCTGAGG GAGAAGGCCA AGGAGCTGTG GC -#AGAGCATC 720 - - TATAACTTGG AGGCAGAGAA GTTCGACCTG CAGGAGAAGT TCAAGCAGCA GA -#AATATGAG 780 - - ATCAATGTTC TCCGAAACAG GATCAACGAT AACCAGAAAG TCTCCAAGAC CC -#GCGGGAAG 840 - - GCTAAAGTCA CCGGGCGCTG GAAATAG - # - # 867 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 651 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA (A) DESCRIPTION: native - #form - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - ATGGCTAGCA TGGGATCTAT GGCGGATGGG AGCAGCGATG CGGCTAGGGA AC -#CTCGCCCT 60 - - GCACCAGCCC CAATCAGACG CCGCTCCTCC AACTACCGCG CTTATGCCAC GG -#AGCCGCAC 120 - - GCCAAGAAAA AATCTAAGAT CTCCGCCTCG AGAAAATTGC AGCTGAAGAC TC -#TGCTGCTG 180 - - CAGATTGCAA AGCAAGAGCT GGAGCGAGAG GCGGAGGAGC GGCGCGGAGA GA -#AGGGGCGC 240 - - GCTCTGAGCA CCCGCTGCCA GCCGCTGGAG TTGGCCGGGC TGGGCTTCGC GG -#AGCTGCAG 300 - - GACTTGTGCC GACAGCTCCA CGCCCGTGTG GACAAGGTGG ATGAAGAGAG AT -#ACGACATA 360 - - GAGGCAAAAG TCACCAAGAA CATCACGGAG ATTGCAGATC TGACTCAGAA GA -#TCTTTGAC 420 - - CTTCGAGGCA AGTTTAAGCG GCCCACCCTG CGGAGAGTGA GGATCTCTGC AG -#ATGCCATG 480 - - ATGCAGGCGC TGCTGGGGGC CCGGGCTAAG GAGTCCCTGG ACCTGCGGGC CC -#ACCTCAAG 540 - - CAGGTGAAGA AGGAGGACAC CGAGAAGGAA AACCGGGAGG TGGGAGACTG GC -#GCAAGAAC 600 - - ATCGATGCAC TGAGTGGAAT GGAGGGCCGC AAGAAAAAGT TTGAGAGCTG A - # 651 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 867 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA (A) DESCRIPTION: native - #form - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - ATGTCTGACA TAGAAGAGGT GGTGGAAGAG TACGAGGAGG AGGAGCAGGA AG -#AAGCAGCT 60 - - GTTGAAGAGC AGGAGGAGGC AGCGGAAGAG GATGCTGAAG CAGAGGCTGA GA -#CCGAGGAG 120 - - ACCAGGGCAG AAGAAGATGA AGAAGAAGAG GAAGCAAAGG AGGCTGAAGA TG -#GCCCAATG 180 - - GAGGAGTCCA AACCAAAGCC CAGGTCGTTC ATGCCCAACT TGGTGCCTCC CA -#AGATCCCC 240 - - GATGGAGAGA GAGTGGACTT TGATGACATC CACCGGAAGC GCATGGAGAA GG -#ACCTGAAT 300 - - GAGTTGCAGG CGCTGATTGA GGCTCACTTT GAGAACAGGA AGAAAGAGGA GG -#AGGAGCTC 360 - - GTTTCTCTCA AAGACAGGAT CGAGAGACGT CGGGCAGAGC GGGCCGAGCA GC -#AGCGCATC 420 - - CGGAATGAGC GGGAGAAGGA GCGGCAGAAC CGCCTGGCTG AAGAGAGGGC TC -#GACGAGAG 480 - - GAGGAGGAGA ACAGGAGGAA GGCTGAGGAT GAGGCCCGGA AGAAGAAGGC TT -#TGTCCAAC 540 - - ATGATGCATT TTGGGGGTTA CATCCAGAAG CAGGCCCAGA CAGAGCGGAA AA -#GTGGGAAG 600 - - AGGCAGACTG AGCGGGAAAA GAAGAAGAAG ATTCTGGCTG AGAGGAGGAA GG -#TGCTGGCC 660 - - ATTGACCACC TGAATGAAGA TCAGCTGAGG GAGAAGGCCA AGGAGCTGTG GC -#AGAGCATC 720 - - TATAACTTGG AGGCAGAGAA GTTCGACCTG CAGGAGAAGT TCAAGCAGCA GA -#AATATGAG 780 - - ATCAATGTTC TCCGAAACAG GATCAACGAT AACCAGAAAG TCTCCAAGAC CC -#GCGGGAAG 840 - - GCTAAAGTCA CCGGGCGCTG GAAATAG - # - # 867 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 216 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - Met Ala Ser Met Gly Ser Met Ala Asp Gly Se - #r Ser Asp Ala Ala Arg 1 5 - # 10 - # 15 - - Glu Pro Arg Pro Ala Pro Ala Pro Ile Arg Ar - #g Arg Ser Ser Asn Tyr 20 - # 25 - # 30 - - Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Ly - #s Lys Ser Lys Ile Ser 35 - # 40 - # 45 - - Ala Ser Arg Lys Leu Gln Leu Lys Thr Leu Le - #u Leu Gln Ile Ala Lys 50 - # 55 - # 60 - - Gln Glu Leu Glu Arg Glu Ala Glu Glu Arg Ar - #g Gly Glu Lys Gly Arg 65 - #70 - #75 - #80 - - Ala Leu Ser Thr Arg Cys Gln Pro Leu Glu Le - #u Ala Gly Leu Gly Phe 85 - # 90 - # 95 - - Ala Glu Leu Gln Asp Leu Cys Arg Gln Leu Hi - #s Ala Arg Val Asp Lys 100 - # 105 - # 110 - - Val Asp Glu Glu Arg Tyr Asp Ile Glu Ala Ly - #s Val Thr Lys Asn Ile 115 - # 120 - # 125 - - Thr Glu Ile Ala Asp Leu Thr Gln Lys Ile Ph - #e Asp Leu Arg Gly Lys 130 - # 135 - # 140 - - Phe Lys Arg Pro Thr Leu Arg Arg Val Arg Il - #e Ser Ala Asp Ala Met 145 1 - #50 1 - #55 1 -#60 - - Met Gln Ala Leu Leu Gly Ala Arg Ala Lys Gl - #u Ser Leu Asp LeuArg 165 - # 170 - # 175 - - Ala His Leu Lys Gln Val Lys Lys Glu Asp Th - #r Glu Lys Glu Asn Arg 180 - # 185 - # 190 - - Glu Val Gly Asp Trp Arg Lys Asn Ile Asp Al - #a Leu Ser Gly Met Glu 195 - # 200 - # 205 - - Gly Arg Lys Lys Lys Phe Glu Ser 210 - # 215 - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 288 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (iii) HYPOTHETICAL: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - Met Ser Asp Ile Glu Glu Val Val Glu Glu Ty - #r Glu Glu Glu Glu Gln 1 5 - # 10 - # 15 - - Glu Glu Ala Ala Val Glu Glu Gln Glu Glu Al - #a Ala Glu Glu Asp Ala 20 - # 25 - # 30 - - Glu Ala Glu Ala Glu Thr Glu Glu Thr Arg Al - #a Glu Glu Asp Glu Glu 35 - # 40 - # 45 - - Glu Glu Glu Ala Lys Glu Ala Glu Asp Gly Pr - #o Met Glu Glu Ser Lys 50 - # 55 - # 60 - - Pro Lys Pro Arg Ser Phe Met Pro Asn Leu Va - #l Pro Pro Lys Ile Pro 65 - #70 - #75 - #80 - - Asp Gly Glu Arg Val Asp Phe Asp Asp Ile Hi - #s Arg Lys Arg Met Glu 85 - # 90 - # 95 - - Lys Asp Leu Asn Glu Leu Gln Ala Leu Ile Gl - #u Ala His Phe Glu Asn 100 - # 105 - # 110 - - Arg Lys Lys Glu Glu Glu Glu Leu Val Ser Le - #u Lys Asp Arg Ile Glu 115 - # 120 - # 125 - - Arg Arg Arg Ala Glu Arg Ala Glu Gln Gln Ar - #g Ile Arg Asn Glu Arg 130 - # 135 - # 140 - - Glu Lys Glu Arg Gln Asn Arg Leu Ala Glu Gl - #u Arg Ala Arg Arg Glu 145 1 - #50 1 - #55 1 -#60 - - Glu Glu Glu Asn Arg Arg Lys Ala Glu Asp Gl - #u Ala Arg Lys LysLys 165 - # 170 - # 175 - - Ala Leu Ser Asn Met Met His Phe Gly Gly Ty - #r Ile Gln Lys Gln Ala 180 - # 185 - # 190 - - Gln Thr Glu Arg Lys Ser Gly Lys Arg Gln Th - #r Glu Arg Glu Lys Lys 195 - # 200 - # 205 - - Lys Lys Ile Leu Ala Glu Arg Arg Lys Val Le - #u Ala Ile Asp His Leu 210 - # 215 - # 220 - - Asn Glu Asp Gln Leu Arg Glu Lys Ala Lys Gl - #u Leu Trp Gln Ser Ile 225 2 - #30 2 - #35 2 -#40 - - Tyr Asn Leu Glu Ala Glu Lys Phe Asp Leu Gl - #n Glu Lys Phe LysGln 245 - # 250 - # 255 - - Gln Lys Tyr Glu Ile Asn Val Leu Arg Asn Ar - #g Ile Asn Asp Asn Gln 260 - # 265 - # 270 - - Lys Val Ser Lys Thr Arg Gly Lys Ala Lys Va - #l Thr Gly Arg Trp Lys 275 - # 280 - # 285__________________________________________________________________________

Number	Name	Date
4946778	Ladner et al.	Aug 1990
5290678	Jackowski	Mar 1994
5560937	Lee et al.	Oct 1996
5583200	Larue et al.	Dec 1996
5604105	Jackowski	Feb 1997

Stable troponin subunits and complexes

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