WO1996012007A1 - Stable recombinant hcmv protease - Google Patents
Stable recombinant hcmv protease Download PDFInfo
- Publication number
- WO1996012007A1 WO1996012007A1 PCT/US1995/012987 US9512987W WO9612007A1 WO 1996012007 A1 WO1996012007 A1 WO 1996012007A1 US 9512987 W US9512987 W US 9512987W WO 9612007 A1 WO9612007 A1 WO 9612007A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hcmv
- protease
- dna
- recombinant
- pcr
- Prior art date
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
- G01N2333/04—Varicella-zoster virus
- G01N2333/045—Cytomegalovirus
Definitions
- HCMV human cytomegalovirus
- the HCMV encodes a protease that participates in the maturation of the viral capsid.
- the enzyme processes the viral assembly protein within the capsid core by mediating cleavage between the ala-ser peptide bond at residue positions 308/309. This results in the linked extrusion of the assembly protein and the encapsidation of the viral genomic DNA.
- the association of the individual assembly proteins into the capsid likely results from specific intermolecular protein interactions.
- the presence of the protease at the N-terminus of a 80kD precursor that also contains the assembly protein assures localization of the enzyme in the capsid as a consequence of interactions mediated by the assembly protein portion.
- a mutant of the herpes simplex virus type 1 (HSV-1 ) which expresses temperature-sensitive alterations in the protease, is incapable of processing the assembly protein and encapsidating genomic DNA at the non-permissive temperature. This result indicates that a specific potent inhibitor of the viral enzyme would be useful as a therapeutic agent.
- V 141G, V207G and V207G were discovered that V 141G, V207G and V207G.
- V254G substitutions in the HCMV protease increase stability.
- the resulting stable recombinant HCMV protease is useful as a screening tool for HCMV antivirals in the potency range of about 100-200 nM, as well as a diagnostic tool for diseases resulting from HCMV infection.
- HCMV cytomegalovirus
- substitutions are glycine for valine at position 141 , glycine for valine at position 207, or glycine for valine at position 254.
- recombinant HCMV protease is useful as a screening tool for HCMV antivirals, as well as a diagnostic tool for diseases resulting from HCMV infection.
- One embodiment of the present invention are the sequences for stable HCMV protease with the double substitution V 141G and V207G (SEQ. ID NOs: 5 & 6).
- One utility for stable HCMV proteases is a screening assay for the detection of compounds that inhibit HCMV protease. This assay has a procedure comprising the steps of:
- Also encompassed in the present invention are compounds that substantially inhibit the stable HCMV protease, i.e., those with an IC 50 value of ⁇ 200 nM.
- Such cells act as hosts and include
- E. coli, B. subtilis, yeasts, fungi, plant cells or animal cells E. coli, B. subtilis, yeasts, fungi, plant cells or animal cells. Expression vectors for many of these host cells have been isolated and
- expression vector for the expression of a desired foreign DNA largely depends on availability of the host cell and how fastidious it is, whether the host cell will support the replication of the expression vector, and other factors readily appreciated by those of ordinary skill in the art.
- the technology for recombinant procaryotic expression systems is now old and conventional.
- the typical host cell is E. coli.
- the technology is illustrated by treatises such as Wu, R (ed) Meth.
- the foreign DNA insert of interest comprises and DNA sequence coding for HCMV (or stable mutant thereof) of the present invention, including any synthetic sequence with this coding capacity or any such cloned sequence or combination thereof.
- HCMV peptides coded and expressed by an entirely recombinant DNA sequence is encompassed by this invention.
- ribosome binding sites appropriate ribosome binding sites, termination codons, enhancers, terminators, or replicon elements.
- additional features can be inserted into the vector at the appropriate site or sites by conventional splicing techniques such as restriction endonuclease digestion and ligation.
- Yeast expression systems which are one variety of recombinant eukaryotic expression systems, generally employ
- Saccharomyces cerevisiae as the species of choice for expressing recombinant proteins.
- S. cerevisiae and similar yeasts possess well known promoters useful in the construction of yeast expression systems, including but not limited to GAP491 , GAL10, ADH2, and alpha mating factor.
- Yeast vectors useful for constructing recombinant yeast expression systems for expressing HCMV include, but are not limited to, shuttle vectors, cosmids, chimeric plasmids, and those having sequences derived from 2-micron circle plasmids.
- Insertion of the appropriate DNA sequence coding for HCMV or stable mutant thereof, into these vectors will, in principle, result in a useful recombinant yeast expression system for HCMV where the modified vector is inserted into the appropriate host cell, by transformation or other means.
- One preferred expression system is with baculovirus, under the control of the polyhedrin promoter. See. e.g., D.R. O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual W.H. Freeman 1992, for a background description of this expression technology.
- a host mammalian cell can be any cell that has been efficiently cloned in cell culture.
- Host mammalian cells useful for the purposes of constructing a recombinant mammalian expression system include, but are not limited to, Vero cells, NIH3T3, GH3, COS, murine Cl27 or mouse L cells.
- Mammalian expression vectors can be based on virus vectors, plasmid vectors which may have SV40, BPV or other viral replicons, or vectors without a replicon for animal cells. Detailed discussions on mammalian expression vectors can be found in the treatises of Glover, D.M. (ed.) "DNA Cloning: A Practical
- Recombinant HCMV may possess additional and desirable structural modifications not shared with the same organically
- synthesized peptide such as adenylation, carboxylation, glycosylation, hydroxylation, methylation, phosphorylation or myristoylation.
- synthesized peptide such as adenylation, carboxylation, glycosylation, hydroxylation, methylation, phosphorylation or myristoylation.
- PCR polymerase chain reaction
- the primer template complexes act as substrate for DNA polymerase which, in performing its replication function, extends the primers.
- Taq DNA Polymerase catalyzes primer extension in the amplification process.
- the enzyme is a thermostable DNA polymerase isolated from Thermus aquaticus. Because it stays active through repeated elevations to high denaturation temperatures, it needs to be added only once. Deoxynucleotide triphosphates provide the building blocks for primer extension.
- telomere sequences are generated as the cycle is repeated.
- the coding domain and any additional primer-encoded information such as restriction sites or translation signals (signal sequences, start codons and/or stop codons) is obtained.
- PCR protocols are often performed at the 100 ⁇ L scale in 0.5 ml microcentrifuge tubes.
- the PCR sample may be single- or double-stranded DNA or RNA. If the starting material is RN A, reverse transcriptase is used to prepare first strand cDNA prior to PCR.
- PCR primers are oligonucleotides, typically 15 to 50 bases long, and are complementary to sequences defining the 5' ends of the complementary template strands. Non-template complementary 5' extensions may be added to primers to allow a variety of useful post amplification operations on the PCR product without significant perturbation of the amplification itself. It is important that the two PCR primers not contain more than two bases complementary with each other, especially at their 3' ends. Internal secondary structure should be avoided in primers.
- PCR buffer is composed of about 50 mM potassium chloride, 10.0 mM Tris-HCl (pH 8.3 at room temperature), 1.5 mM magnesium chloride, and 0.001 % w/v gelatin.
- a titration series in small increments over the 1.5- to 4-mM range will locate the magnesium concentration producing the highest yield of a specific product. Too little free magnesium will result in no PCR product and too much free magnesium may produce a variety of unwanted products.
- a typical PCR protocol for amplification of the DNA template includes an initial 8 minute 94°C denaturation step, followed by 30 cycles of 30 seconds at 94°C
- Primer annealing is usually performed first at 55°C, and the specificity of the product is evaluated. If unwanted bands are observed, the annealing temperature should be raised in subsequent optimization runs. While the primer annealing temperature range is often 37-55°C, it may be raised as high as the extension temperature in some cases. Merging of the primer annealing and primer extension steps results in a two-step PCR process. Primer extension, in most applications, occurs effectively at a temperature of 72°C and seldom needs optimization. In the two-temperature PCR process the temperature range may be 65-70°C. In situations where enzyme concentration limits amplification in late cycles, the extension is preferably increased linearly with cyclic number. Usually, 25 to 45 cycles are required for extensive
- amplification i.e., 1 ,000,000 fold of a specific target.
- HCMV strain AD 169 DNA was prepared from supernate virions as previously described [LaFemina, R.L., et al., J. Gen Vir., 64. 373 ( 1983)].
- the N-terminal 256 amino acid protease domain was PCR amplified using primers derived from the DNA seguence as described by Chee, M.S., et al., Curr. Top Microbiol. Immunol., 154, 125 ( 1990), Genbank accession number X17403.
- the seguence of the N-terminal primer was 5'GCTAGGCTCATATGACGATGGACGAGCAGCAG (SEQ ID NO: 7), while the seguence of the C-terminal primer was 5'GCTAGGCTAGATCTTTACGCCTTGACGTATGACTCGC (SEQ ID NO: 8).
- PCR conditions consisted of: 6 cycles with 0.5 min
- the amplified DNA was digested with Ndel and Bglll prior to ligation into the Ndel and BamHI sites of the T7 expression vector pET3c.
- the resulting plasmid, pT7CMVPr-4, was introduced into E. coli BL21 DE3 for expression. Expression was induced by standard IPTG induction for 2 hr.
- microfluidizer at 25 psi.
- the lysate (20 ml) was centrifuged 30 min at 8,000 ⁇ g and the pellet washed twice with 10 ml of lysis buffer containing 0.1 % NP40.
- the pellet was incubated at 40°C under N 2 in 4.5 ml of 6.0
- the enzyme was loaded in Buffer A (25 mM Tris-HCl, pH 7.5, 1 mM DTT and 1 mM EDTA) at 0.3 ml/min and eluted with a 0-1.0 M NaCl salt gradient at 1 ml/min.
- the gradient used was Buffer A for 10 min, linear increase to 0.3 M NaCl in Buffer A for 30 min, followed by linear increase to 1.0 M NaCl in 15 min.
- the active protease eluted at 0.15 to 0.20 M NaCl.
- the fractions were analyzed by SDS-PAGE and by native gel electrophoresis as well as by two HPLC sizing columns attached in series (300 ⁇ 7.5 mm) in PBS.
- Absorbance of the eluate was monitored at 210 nm using a photodiode array detector.
- the enzyme concentration used in the assay varied from 150 to 1500 nM depending on the substrate used.
- Each substrate peptide was titrated from 50 ⁇ M to 5.0 mM.
- Kinetic parameters (kcat and km) were determined by fitting the velocity (initial rates at ⁇ 5.0% of total substrate hydrolysis) versus substrate concentration data to the Michaelis-Menton equation (hyperbolic). The initial velocity and steady-state conditions for the enzyme reaction were established for each peptide substrate.
- the recombinant human cytomegalovirus protease (with V 141G and V207G substitutions, 256 amino acids, 20 nM) cleaves the peptide substrate 3 H-Acetyl Gly Val Val Asn Ala Ser Cys Arg Leu Arg Arg amide ( 1 mM) (SEQ ID NO. 9) at the Ala Ser bond.
- the assay is performed in 100 mM Hepes (pH 7.5), 1 mM EDTA .05% BSA, 25 mM NaCl (50 ⁇ l total volume) and quenched by adding 50 ⁇ l of 5% phosphoric acid.
- the assay mix is transferred to a tube containing Dowex ion exchange, the tubes are rinsed with water (2 ⁇ 200 ⁇ l).
- the cleaved radioactive peptide in the supernatant is quantitated by a scintillation counter. Reduction in radioactivity in presence of compounds gives the measure of inhibition, and is determined as the concentration of inhibitory compound giving 50% inhibition or IC 50 .
- Highly potent compounds generally have IC 50 less than or equal to about 200 nM.
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Abstract
The protease of human cytomegalovirus (HCMV) is rendered stable by certain mutations discovered by applicants, and is useful as a screening tool for HCMV antivirals as well as a diagnostic tool for diseases resulting from HCMV infection.
Description
TITLE OF THE INVENTION
STABLE RECOMBINANT HCMV PROTEASE
BACKGROUND OF THE INVENTION
The human cytomegalovirus (HCMV) is the etiological agent of a variety of infectious diseases in infants and children. The virus also is involved in severe infections of adults with
immunodeficiencies, such as AIDS patients or transplant recipients. The HCMV encodes a protease that participates in the maturation of the viral capsid. The enzyme processes the viral assembly protein within the capsid core by mediating cleavage between the ala-ser peptide bond at residue positions 308/309. This results in the linked extrusion of the assembly protein and the encapsidation of the viral genomic DNA.
The association of the individual assembly proteins into the capsid likely results from specific intermolecular protein interactions. The presence of the protease at the N-terminus of a 80kD precursor that also contains the assembly protein assures localization of the enzyme in the capsid as a consequence of interactions mediated by the assembly protein portion. A mutant of the herpes simplex virus type 1 (HSV-1 ), which expresses temperature-sensitive alterations in the protease, is incapable of processing the assembly protein and encapsidating genomic DNA at the non-permissive temperature. This result indicates that a specific potent inhibitor of the viral enzyme would be useful as a therapeutic agent.
Applicants have discovered that V 141G, V207G and
V254G substitutions in the HCMV protease increase stability. The resulting stable recombinant HCMV protease is useful as a screening tool for HCMV antivirals in the potency range of about 100-200 nM, as well as a diagnostic tool for diseases resulting from HCMV infection.
SUMMARY OF THE INVENTION
Certain point mutations created by site-directed mutagenesis are disclosed to increase the stability of human
cytomegalovirus (HCMV) protease. The resulting amino acid
substitutions are glycine for valine at position 141 , glycine for valine at position 207, or glycine for valine at position 254. The stable
recombinant HCMV protease is useful as a screening tool for HCMV antivirals, as well as a diagnostic tool for diseases resulting from HCMV infection.
DETAILED DESCRIPTION OF THE INVENTION
There is disclosed the sequences for stable HCMV protease with either a V 141G substitution (SEQ. ID NOs: 3 & 4), or the V207G substitution or the V254G substitution, or combination thereof. Such proteases are stable to autocatalysis, and are useful as a screening tool for HCMV antivirals, as well as a diagnostic tool for diseases resulting from HCMV infection.
One embodiment of the present invention are the sequences for stable HCMV protease with the double substitution V 141G and V207G (SEQ. ID NOs: 5 & 6).
Also disclosed are DNA vectors comprising a DNA sequence for stable HCMV proteases having these substitutions, as well as recombinant expression systems for expression.
One utility for stable HCMV proteases is a screening assay for the detection of compounds that inhibit HCMV protease. This assay has a procedure comprising the steps of:
(a) providing a quantity of a compound or compounds to be assayed;
(b) incubating said compound or compounds with a
stable HCMV protease having the substitutions selected from V 141G, V207G or V254G or combination thereof;
(c) determining the inhibition of said protease in a
substrate cleavage assay.
Also encompassed in the present invention are compounds that substantially inhibit the stable HCMV protease, i.e., those with an IC50 value of≤ 200 nM.
Expression of HCMV Protease in a Recombinant Expression System
It is now a relatively straightforward technology to prepare cells expressing a foreign gene. Such cells act as hosts and include
E. coli, B. subtilis, yeasts, fungi, plant cells or animal cells. Expression vectors for many of these host cells have been isolated and
characterized, and are used as starting materials in the construction, through conventional recombinant DNA techniques, of vectors having a foreign DNA insert of interest. Any DNA is foreign if it does not naturally derive from the host cells used to express the DNA insert. The foreign DNA insert may be expressed on extrachromosomal plasmids or after integration in whole or in part in the host cell chromosome(s), or may actually exist in the host cell as a combination of more than one molecular form. The choice of host cell and
expression vector for the expression of a desired foreign DNA largely depends on availability of the host cell and how fastidious it is, whether the host cell will support the replication of the expression vector, and other factors readily appreciated by those of ordinary skill in the art.
The technology for recombinant procaryotic expression systems is now old and conventional. The typical host cell is E. coli. The technology is illustrated by treatises such as Wu, R (ed) Meth.
Enzymol., 68 (1979) and Maniatis, T. et al., Molecular Cloning: A Laboratory Manual Cold Spring Harbor 1982.
The foreign DNA insert of interest comprises and DNA sequence coding for HCMV (or stable mutant thereof) of the present invention, including any synthetic sequence with this coding capacity or any such cloned sequence or combination thereof. For example, HCMV
peptides coded and expressed by an entirely recombinant DNA sequence is encompassed by this invention.
Vectors useful for constructing eukaryotic expression systems for the production of recombinant HCMV comprise the DNA sequence for HCMV or variant thereof, operatively linked thereto with appropriate transcriptional activation DNA sequences, such as a promoter and/or operator. Other typical features may include
appropriate ribosome binding sites, termination codons, enhancers, terminators, or replicon elements. These additional features can be inserted into the vector at the appropriate site or sites by conventional splicing techniques such as restriction endonuclease digestion and ligation.
Yeast expression systems, which are one variety of recombinant eukaryotic expression systems, generally employ
Saccharomyces cerevisiae as the species of choice for expressing recombinant proteins. S. cerevisiae and similar yeasts possess well known promoters useful in the construction of yeast expression systems, including but not limited to GAP491 , GAL10, ADH2, and alpha mating factor.
Yeast vectors useful for constructing recombinant yeast expression systems for expressing HCMV include, but are not limited to, shuttle vectors, cosmids, chimeric plasmids, and those having sequences derived from 2-micron circle plasmids.
Insertion of the appropriate DNA sequence coding for HCMV or stable mutant thereof, into these vectors will, in principle, result in a useful recombinant yeast expression system for HCMV where the modified vector is inserted into the appropriate host cell, by transformation or other means.
One preferred expression system is with baculovirus, under the control of the polyhedrin promoter. See. e.g., D.R. O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual W.H. Freeman 1992, for a background description of this expression technology.
Recombinant mammalian expression systems are another means of producing the recombinant HCMV for the conjugates of this
invention. In general, a host mammalian cell can be any cell that has been efficiently cloned in cell culture. Host mammalian cells useful for the purposes of constructing a recombinant mammalian expression system include, but are not limited to, Vero cells, NIH3T3, GH3, COS, murine Cl27 or mouse L cells. Mammalian expression vectors can be based on virus vectors, plasmid vectors which may have SV40, BPV or other viral replicons, or vectors without a replicon for animal cells. Detailed discussions on mammalian expression vectors can be found in the treatises of Glover, D.M. (ed.) "DNA Cloning: A Practical
Approach," IRL 1985, Vols. I and II.
Recombinant HCMV may possess additional and desirable structural modifications not shared with the same organically
synthesized peptide, such as adenylation, carboxylation, glycosylation, hydroxylation, methylation, phosphorylation or myristoylation. These added features may be chosen or preferred as the case may be, by the appropriate choice of recombinant expression system. On the other hand, recombinant HCMV may have its sequence extended by the principles and practice of organic synthesis. Polymerase Chain Reaction Amplification
Large amounts of DNA coding for HCMV protein may be obtained using polymerase chain reaction (PCR) amplification
techniques as described in Mullins et al., U.S. Patent No. 4,800,159 and other published sources. See also, for example, Innis, M.A. et al., PCR Protocols Academic, 1990, especially chapter 22 therein, which is Higuchi, R., "Recombinant PCR." The extension product of one primer, when hybridized to another primer, becomes a template for the synthesis of another nucleic acid molecule.
The primer template complexes act as substrate for DNA polymerase which, in performing its replication function, extends the primers. The region in common with both primer extensions, upon denaturation, serves as template for a repeated primer extension.
Taq DNA Polymerase catalyzes primer extension in the amplification process. The enzyme is a thermostable DNA polymerase
isolated from Thermus aquaticus. Because it stays active through repeated elevations to high denaturation temperatures, it needs to be added only once. Deoxynucleotide triphosphates provide the building blocks for primer extension.
The nucleic acid sequence strands are heated until they separate, in the presence of oligonucleotide primers that bind to their complementary strand at a particular site on the template. This process is continued with a series of heating and cooling cycles, heating to separate strands, and cooling to reanneal and extend the sequences.
More and more copies of the strands are generated as the cycle is repeated. Through amplification, the coding domain and any additional primer-encoded information such as restriction sites or translation signals (signal sequences, start codons and/or stop codons) is obtained. PCR protocols are often performed at the 100 μL scale in 0.5 ml microcentrifuge tubes. The PCR sample may be single- or double-stranded DNA or RNA. If the starting material is RN A, reverse transcriptase is used to prepare first strand cDNA prior to PCR.
Typically, nanogram amounts of cloned template, up to microgram amounts of genomic DNA, or 20,000 target copies are chosen to start optimization trials.
PCR primers are oligonucleotides, typically 15 to 50 bases long, and are complementary to sequences defining the 5' ends of the complementary template strands. Non-template complementary 5' extensions may be added to primers to allow a variety of useful post amplification operations on the PCR product without significant perturbation of the amplification itself. It is important that the two PCR primers not contain more than two bases complementary with each other, especially at their 3' ends. Internal secondary structure should be avoided in primers.
Because Taq DNA Polymerase has activity in the 37-55°C range, primer extension will occur during the annealing step and the hybrid will be stabilized. The concentrations of the primers are preferably equal in conventional PCR and, typically, are in vast excess of the template to be reproduced.
In one typical PCR protocol, each deoxynucleotide triphosphate concentration is preferably about 200 μM. The four dNTP concentrations are preferably above the estimated Km of each dNTP ( 10-15 μM).
Preferably PCR buffer is composed of about 50 mM potassium chloride, 10.0 mM Tris-HCl (pH 8.3 at room temperature), 1.5 mM magnesium chloride, and 0.001 % w/v gelatin. In the presence of 0.8 mM total dNTP concentration, a titration series in small increments over the 1.5- to 4-mM range will locate the magnesium concentration producing the highest yield of a specific product. Too little free magnesium will result in no PCR product and too much free magnesium may produce a variety of unwanted products.
Preferably, in a 100- μL reaction volume, 2.0 to 2.5 units of Taq DNA Polymerase are recommended. The enzyme can be added conveniently to a fresh master mix prepared for a number of reactions, thereby avoiding accuracy problems associated with adding individual 0.5- μL enzyme aliquots to each tube. A typical PCR protocol for amplification of the DNA template includes an initial 8 minute 94°C denaturation step, followed by 30 cycles of 30 seconds at 94°C
(denaturation), 1 minute at 55°C (primer annealing), and 2 minutes at 72°C (polymerization). At the end of the last cycle, all strands are completed by a 5 minute incubation at 72°C.
During DNA denaturation, sufficient time must be allowed for thermal equilibration of the sample. The practical range of effective denaturation temperatures for most samples is 92-95°C, with 94°C being the standard choice.
Primer annealing is usually performed first at 55°C, and the specificity of the product is evaluated. If unwanted bands are observed, the annealing temperature should be raised in subsequent optimization runs. While the primer annealing temperature range is often 37-55°C, it may be raised as high as the extension temperature in some cases. Merging of the primer annealing and primer extension steps results in a two-step PCR process.
Primer extension, in most applications, occurs effectively at a temperature of 72°C and seldom needs optimization. In the two-temperature PCR process the temperature range may be 65-70°C. In situations where enzyme concentration limits amplification in late cycles, the extension is preferably increased linearly with cyclic number. Usually, 25 to 45 cycles are required for extensive
amplification (i.e., 1 ,000,000 fold) of a specific target.
Once the DNA sequence is determined, through conventional and well-known techniques, its amino acid sequence can be deduced by "translating" the DNA seguence. The resulting amino acid seguence having the selected HCMV seguence is then employed to synthesize large guantities of HCMV protein. Synthesis is performed by organic synthesis or by recombinant expression systems, or both. EXAMPLE 1
Cloning and Expression of the HCMV Protease
HCMV strain AD 169 DNA was prepared from supernate virions as previously described [LaFemina, R.L., et al., J. Gen Vir., 64. 373 ( 1983)]. The N-terminal 256 amino acid protease domain was PCR amplified using primers derived from the DNA seguence as described by Chee, M.S., et al., Curr. Top Microbiol. Immunol., 154, 125 ( 1990), Genbank accession number X17403. The seguence of the N-terminal primer was 5'GCTAGGCTCATATGACGATGGACGAGCAGCAG (SEQ ID NO: 7), while the seguence of the C-terminal primer was 5'GCTAGGCTAGATCTTTACGCCTTGACGTATGACTCGC (SEQ ID NO: 8). PCR conditions consisted of: 6 cycles with 0.5 min
denaturation at 97°C, 1.5 min annealing at 60°C and 2 min extension at 72°C, 25 cycles with 1 min denaturation at 94°C and annealing and extension as above; followed by 6 cycles with 1 min denaturation at 94°C, 1.5 min annealing at 60°C and 4 min extension at 72°C. The amplified DNA was digested with Ndel and Bglll prior to ligation into the Ndel and BamHI sites of the T7 expression vector pET3c. The resulting plasmid, pT7CMVPr-4, was introduced into E. coli BL21 DE3
for expression. Expression was induced by standard IPTG induction for 2 hr.
EXAMPLE 2
Purification and Refolding of Recombinant HCMV Protease
Cells from 450 ml of induced cultures were collected by centrifugation and resuspended in 15 ml of 50 mM Tris-HCl, pH 7.8, 25 mM NaCl, 1 mM EDTA, 1 mM DTT and 10% glycerol (Lysis buffer) at 4°C. Phenylmethylsulfonyl fluoride was added to a concentration of 1 mM, and the cells were disrupted by two passes through a
microfluidizer at 25 psi. The lysate (20 ml) was centrifuged 30 min at 8,000 ×g and the pellet washed twice with 10 ml of lysis buffer containing 0.1 % NP40.
The pellet was incubated at 40°C under N2 in 4.5 ml of 6.0
M guanidine HCl, 100 mM Tris-HCl, pH 8.2, 50 mM NaCl, 1 mM EDTA and 200 mM β-mercaptoethanol for 45 min and a second aliguot of 100 mM β-mercaptoethanol was added and incubation continued for 45 min. The solubilized reduced protease was diluted to approximately 100 μg/ml (450 ml) in 1.0 M guanidine HCl, 25 mM Tris-HCl, pH 7.8, 25 mM NaCl. 1 mM EDTA, 2 mM reduced glutathione, and 0.2 mM oxidized glutathione and dialyzed at 4°C for 18-20 hr against the same buffer. Further dialysis was done for 48 hrs with 6 × 4 liters of 25 mM Tris-HCl, pH 7.5, 25 mM NaCl, 1 mM DTT and 1 mM EDTA. The refolded solubilized protease was concentrated on a 150 ml disposable stirred cell filtration device and fractionated on an anion exchange MonoQ fast performance liquid chromatography analytical column (0.5 × 5 cm). The enzyme was loaded in Buffer A (25 mM Tris-HCl, pH 7.5, 1 mM DTT and 1 mM EDTA) at 0.3 ml/min and eluted with a 0-1.0 M NaCl salt gradient at 1 ml/min. The gradient used was Buffer A for 10 min, linear increase to 0.3 M NaCl in Buffer A for 30 min, followed by linear increase to 1.0 M NaCl in 15 min. The active protease eluted at 0.15 to 0.20 M NaCl. The fractions were analyzed by SDS-PAGE and by native gel electrophoresis as well as by two HPLC
sizing columns attached in series (300 × 7.5 mm) in PBS. Activity of the protease was followed by substrate cleavage assay. The >95% pure fractions were pooled and stored at -70°C in the elution buffer containing 5% glycerol. The cleavage activity of enzyme was studied in the pH range 3.0 to 9.5 and the optimum pH was found to be pH 7.5.
EXAMPLE 3
Site-Directed Mutagenesis
See R. Higuchi, "Recombinant PCR," in M.A. Innis, et al.,
(eds.) PCR PROTOCOLS: A Guide to Methods and Applications, Academic, 1990 pp 177-183. The mismatch for the amino acid substitution is placed in the middle of the DNA primer. EXAMPLE 4
Substrate Cleavage Assay
Peptide substrates were organically synthesized with a peptide synthesizer and were >95% pure. The peptide cleavage assay was performed at room temperature in 50 μl of 100.0 mM Hepes buffer (pH 7.5), 5.0 mM DTT, 1.0 mM EDTA, 25.0 mM NaCl, 0.05% bovine serum albumin. After 20 mins, the reaction was quenched by addition of 50 μl 10% phosphoric acid and the mixture was analyzed by reverse phase HPLC on a 3.9 × 75 mm column. The cleavage products were resolved using a 0.1 % phosphoric acid/acetonitrile gradient and identified by either N-terminus sequence analysis or retention time comparison with authentic peptide. Absorbance of the eluate was monitored at 210 nm using a photodiode array detector. The enzyme concentration used in the assay varied from 150 to 1500 nM depending on the substrate used. Each substrate peptide was titrated from 50 μM to 5.0 mM. Kinetic parameters (kcat and km) were determined by fitting the velocity (initial rates at <5.0% of total substrate hydrolysis) versus substrate concentration data to the Michaelis-Menton equation
(hyperbolic). The initial velocity and steady-state conditions for the enzyme reaction were established for each peptide substrate.
EXAMPLE 5
Assay for HCMV Protease Inhibition
The recombinant human cytomegalovirus protease (with V 141G and V207G substitutions, 256 amino acids, 20 nM) cleaves the peptide substrate 3H-Acetyl Gly Val Val Asn Ala Ser Cys Arg Leu Arg Arg amide ( 1 mM) (SEQ ID NO. 9) at the Ala Ser bond. The assay is performed in 100 mM Hepes (pH 7.5), 1 mM EDTA .05% BSA, 25 mM NaCl (50 μl total volume) and quenched by adding 50 μl of 5% phosphoric acid. The assay mix is transferred to a tube containing Dowex ion exchange, the tubes are rinsed with water (2 × 200 μl). The cleaved radioactive peptide in the supernatant is quantitated by a scintillation counter. Reduction in radioactivity in presence of compounds gives the measure of inhibition, and is determined as the concentration of inhibitory compound giving 50% inhibition or IC50. Highly potent compounds generally have IC50 less than or equal to about 200 nM.
While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations, modifications, deletions or additions of procedures and protocols described herein, as come within the scope of the following claims and its equivalents.
Claims
1 . Stable HCMV protease with either V 141 G, V207G or V254G substitutions, or combination thereof.
2. A DNA comprising a DNA sequence coding for the protease of Claim 1.
3. A DNA vector comprising a DNA sequence coding for the protease of Claim 1.
4. An expression system for the expression of the protease of Claim 1.
5. A screening assay for the detection of compounds that inhibit HCMV protease, said assay having a procedure comprising the steps of:
(a) providing a quantity of a compound or compounds to be assayed;
(b) incubating said compound or compounds with the stable HCMV protease of Claim 1 ;
(c) determining the inhibition of said protease in a
substrate cleavage assay.
6. The compounds that inhibit HCMV protease with IC50≤ 200 nM, as measured by the screening assay of Claim 5.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU39528/95A AU3952895A (en) | 1994-10-17 | 1995-10-13 | Stable recombinant hcmv protease |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US32395394A | 1994-10-17 | 1994-10-17 | |
US323,953 | 1994-10-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996012007A1 true WO1996012007A1 (en) | 1996-04-25 |
Family
ID=23261428
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1995/012987 WO1996012007A1 (en) | 1994-10-17 | 1995-10-13 | Stable recombinant hcmv protease |
Country Status (2)
Country | Link |
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AU (1) | AU3952895A (en) |
WO (1) | WO1996012007A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2311605A (en) * | 1996-03-26 | 1997-10-01 | Merck & Co Inc | Assay for inhibitors of human cytomegalovirus (HCMV) protease |
WO1998004286A3 (en) * | 1996-07-26 | 1998-05-14 | Searle & Co | Assembly-deficient herpesvirus vaccine |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5434074A (en) * | 1991-07-05 | 1995-07-18 | Gibson; D. Wade | Cytomegalovirus proteinase |
-
1995
- 1995-10-13 AU AU39528/95A patent/AU3952895A/en not_active Abandoned
- 1995-10-13 WO PCT/US1995/012987 patent/WO1996012007A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5434074A (en) * | 1991-07-05 | 1995-07-18 | Gibson; D. Wade | Cytomegalovirus proteinase |
Non-Patent Citations (2)
Title |
---|
BAUM ET AL: "Expression and Analysis of the Human Cytomegalovirus UL80-Encoded Protease: Identification of Autoproteolytic Sites.", JOURNAL OF VIROLOGY, vol. 67, no. 1, January 1993 (1993-01-01), pages 497 - 506 * |
JONES ET AL: "Proteolytic Activity of Human Cytomegalovirus UL80 Protease Cleavage Site Mutants.", JOURNAL OF VIROLOGY, vol. 68, no. 6, January 1993 (1993-01-01), pages 3742 - 3752 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2311605A (en) * | 1996-03-26 | 1997-10-01 | Merck & Co Inc | Assay for inhibitors of human cytomegalovirus (HCMV) protease |
WO1998004286A3 (en) * | 1996-07-26 | 1998-05-14 | Searle & Co | Assembly-deficient herpesvirus vaccine |
Also Published As
Publication number | Publication date |
---|---|
AU3952895A (en) | 1996-05-06 |
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