3-hydroxy-3-methyl-glutaryl-coa synthase preparation with improved stability

A preparation of recombinant 3-hydroxy-3-methylglutaryl-CoA synthase is disclosed, wherein the preparation has at least 0.024 unitsg specific activity. Preferably, the preparation has at least 0.24 units/mg specific activity and is a crude cell extract. Preferably, at least 90% of the synthase molecules have not been substantially proteolytically cleaved. A preparation of recombinant HMG-CoA synthase wherein the preparation retains 50% activity after storage at 4.degree. C. for six months is also disclosed. A method of evaluating the efficacy of candidate anti-isoprene and anti-cholesterol drugs is also disclosed which comprises exposing the candidate drug to a recombinant 3-hydroxy-3-methylglutaryl CoA synthase preparation.

FIELD OF THE INVENTION 
The present invention relates to enzyme purification and storage. More 
specifically, the present invention relates to a recombinant 
3-hydroxy-3-methylglutaryl-CoA synthase preparation with high specific 
activity that lacks substantial proteolytic cleavage, is stable during 
long-term 4.degree. C. storage and is suitable as a reagent in assays for 
evaluating anti-cholesterol and anti-isoprene agents. 
BACKGROUND 
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (E.C. 4.1.3.5) catalyzes 
the formation of a key intermediate in the cholesterogenic and ketogenic 
pathways in a three step process (Miziorko, et al., (1977) J. Biol. Chem. 
252, 1414-1420): 
(1) EnzSH+acetyl-CoA.fwdarw.acetyl-SEnz+CoASH 
(2) acetoacetyl-CoA+acetyl-SEnz.fwdarw.EnzS-HMG-CoA 
(3) EnzS-HMG-CoA+H.sub.2 O.fwdarw.EnzSH+HMG-CoA 
Distinct hepatic isozymes catalyze the synthesis of cholesterogenic and 
ketogenic intermediates (Clinkenbeard et al., (1975) J. Biol. Chem. 250, 
3124-3135). As anticipated for the enzyme that catalyzes the first 
irreversible step in these metabolic pathways, HMG-CoA synthase has been 
implicated as a control point (Smith et al., (1988) J. Biol. Chem. 263, 
18480-18487; Casals et al., (1992) Biochem. J. 283, 261-264; Quant et al., 
(1989) Biochem. J. 262, 159-164). These observations account for the 
recent interest in developing anti-steroidogenic and anti-isoprenyl agents 
that selectively target this enzyme (Omura et al., (1987) J. Antibiotics 
40, 1356-1357). 
Elements of the active site of this important enzyme have been identified 
(Miziorko, et al. (1985) Biochemistry 24, 3174-3179; Miziorko, et al., 
(1985) J. Biol. Chem. 260, 13513-13516; Vollmer et al., (1988) 
Biochemistry 27, 4288-4292; Miziorko et al., (1990) Biochim. Biophys. Acta 
1041, 273278) in studies that relied on synthase isolated from an avian 
source. These synthase preparations have particular characteristics that 
render their use in drug targeting studies problematic. For example, an 
avian liver cytosolic synthase (Clinkenbeard, et al., 1975, supra) was 
found to be proteolytically cleaved. This would account for the 
differences in molecular weight, isoelectric point, and chromatographic 
properties that were observed for four different putative cytosolic 
protein species that, upon isolation, were found to catalyze the formation 
of HMG-CoA. Miziorko ((1985) Methods of Enzymology 110, p. 19-26, Ed. J. 
H. Law and Hans C. Rilling) noted that a crude preparation of HMG-CoA 
synthase isolated from chicken liver had only 20% activity after storage 
at 4.degree. C. for 24 hours. 
HMG-CoA synthase is used to assay drugs believed to be efficacious in 
cholesterol reduction because of the pivotal role the synthase plays in 
the production of cholesterol. Similarly, one could use HMG-CoA synthase 
preparations to assay drugs thought to be capable of inhibiting 
isoprenylation of proteins, which may be a step in cancer metabolism. 
Investigation of putative anti-cholesterol and anti-isoprene drugs is 
handicapped by the quality of prior art native HMG-CoA synthase 
preparations. Additional investigation of synthase properties could be 
facilitated by application of recombinant DNA methodology to allow more 
convenient production of the enzyme and engineered variants. We previously 
documented the isolation of full length cDNA encoding avian liver HMG-CoA 
synthase (Kattar-Cooley et al., (1990) Arch. Biochem. Biophys. 283, 
523-529). The genes for the rat mitochondrial and cytosolic HMG-CoA 
synthase have been analyzed by Ayte, et al., (1990, Proc. Natl. Acad. Sci. 
U.S.A. 87, 3874-3878). These workers expressed cDNA clones of the synthase 
gene in E. coli with a resulting specific activity of between 0.4 and 1.2 
milliunits/mg. Hamster HMG-CoA synthase was cloned by Gil, et al. (Gil, et 
al., 1986, J. Biol. Chem. 261, 3710-3716.). 
SUMMARY OF THE INVENTION 
The present invention is a preparation of recombinant HMG-CoA synthase with 
improved stability properties. In one embodiment of the invention, the 
preparation has at least 0.024 units/mg specific activity. Preferably, the 
preparation has at least 0.240 units/mg specific activity and is a crude 
cell extract. In a preferred form, at least 90% of the synthase molecules 
have not been substantially proteolytically cleaved. By "substantially 
proteolytically cleaved", we mean that only the initial methionine of the 
protein is cleaved and the rest of the protein is intact. 
In another embodiment, the present invention is a preparation of 
recombinant HMG-CoA synthase retaining 50% activity after storage at 
4.degree. C. for six months. Preferably, this preparation retains 50% 
activity after storage at one year at 4.degree. C. 
In another embodiment, the present invention is a method for analyzing the 
efficacy of anti-cholesterol or anti-isoprene drugs, comprising the step 
of exposing the candidate drug to the preparations of HMG-CoA synthase 
disclosed above. 
It is an advantage of the present invention that an HMG-CoA synthase 
preparation with improved stability qualities is provided. 
Another advantage of the present invention is that a preparation yielding 
sufficient amounts and quality of HMG-CoA synthase to more effectively 
analyze anti-cholesterol and anti-isoprene drugs is enabled. 
Another advantage of the present invention is that a recombinant avian 
HMG-CoA synthase preparation with improved stability characteristics is 
provided. 
Another advantage of the present invention is that a preparation of 
cytosolic HMG-CoA synthase with improved stability characteristics is 
provided. 
It is a feature of the present invention that the HMG-CoA synthase 
preparation is at least 50% active after storage at 4.degree. C. for six 
months. 
It is another feature of the present invention that at least 90% of the 
preparation is not substantially proteolytically cleaved. 
It is another feature of the present invention that a crude bacterial 
lysate extract of the synthase has at least 0.025 units/mg specific 
activity.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
The present invention is a preparation of recombinant HMG-CoA synthase with 
improved stability characteristics. In one embodiment, a crude cell 
extract preparation has a specific activity of at least 0.024 units/mg, 
preferably a specific activity of at least 0.24 units/mg. By "crude cell 
extract" preparation we mean an enzyme preparation that has not been 
substantially purified from the host cell producing the enzyme. Also 
preferably, at least 90% of the molecules have not been substantially 
proteolytically cleaved. In another embodiment, the present invention is 
an HMG-CoA synthase preparation that retains at least 50% activity after 
storage at 4.degree. C. for six months, preferably for one year. 
The preparation of the present invention will be useful in studies 
evaluating drugs as anti-cholesterol and anti-isoprene agents. The 
improved stability of the preparation of the present invention will allow 
workers to perform these studies on a reliable, consistent protein 
preparation and make determination of the candidate drug's efficacy an 
easier task. 
1. Creation of a Recombinant HMG-CoA Synthase Expression System 
A nucleic acid sequence encoding HMG-CoA synthase is obtained and joined to 
appropriate regulatory sequences. A preferable sequence is disclosed in 
SEQ ID NO:1. The Examples below disclose how SEQ ID NO:1 was obtained from 
the avian cDNA clone described in Kattar-Cooley, et al., supra (hereby 
incorporated by reference). The first base (C) in SEQ ID NO:1 is a 
noncoding base added to furnish an Nco I site. The rest of the sequence is 
coding sequence up to nucleotide 1570. The nucleotides between 1571 and 
1824 are noncoding sequences. The resulting sequence described in the 
Examples has the second amino acid (a proline in native HMG-CoA synthase) 
replaced by an alanine. The translation product of the avian cDNA is 
presented in SEQ ID NO:2. 
To replicate SEQ ID NO:1, one would most easily start by obtaining an avian 
HMG-CoA cDNA clone. To obtain an avian HMG-CoA cDNA clone, one could use 
SEQ ID NO:1 to construct probes suitable for screening an avian cDNA 
library. Alternatively, one could construct primers for use in a 
polymerase chain reaction following synthesis of a DNA strand from avian 
messenger RNA using reverse transcription. Other methods will be known to 
one skilled in the art. One could then use method described in the 
Examples to manipulate the cDNA sequence. 
Clones for HMG-CoA synthase have also been isolated from rat (mitochondrial 
and cytosolic), human, and hamster. The translation product for rat 
mitochondrial synthase is SEQ ID NO:3, for hamster synthase is SEQ ID 
NO:4, for rat cytosolic synthase is SEQ ID NO:5, and for human synthase is 
SEQ ID NO:6. (The rat mitochondrial sequence is disclosed in Ayte, et al., 
1990, Natl. Acad. Sci., 87, 3874-3878; the rat cytosolic sequence is 
disclosed in Ayte, et al., 1990, Nucl. Acids Res., 18, 3642-3642; the 
hamster sequence is disclosed in Gil, et al., 1986, J. Biol. Chem., 261, 
3710-3716; and the human sequence is disclosed in Russ, et al., 1992, 
Biochem. Biophys. Acta, 1132, 329-331.) 
FIG. 1 compares the amino acid sequences of the different HMG-CoA synthases 
and demonstrates the homology between them. The sequences in FIG. 1 have 
the following legend: "Hmcs$Crigr" indicates the hamster sequence; 
"Hmcs$Rat" indicates the rat cytosolic sequence; "Synthase" indicates the 
human sequence; "Avcytsyn" indicates the avian sequence and "Hmcm$Rat" 
indicates the rat mitochondrial sequence. In general, a high (&gt;80%) degree 
of homology is observed between cytosolic HMG-CoA synthases. Comparison 
between cytosolic and mitochondrial isozymes shows lower (&lt;70%) homology, 
even when the isozymes are derived from the same tissue. 
Appropriate regulatory sequences must be added to the HMG-CoA synthase 
sequence for protein expression. The Examples below demonstrate suitable 
appropriate sequences for expression in an E. coli host. If another host 
is desired, such as yeast or animal cells, the art is knowledgeable about 
other appropriate regulatory sequences. 
The recombinant protein produced by the host must then be isolated, 
preferably as described in the Examples, although other isolation 
techniques would be equally suitable. The protocol described in the 
Examples is analogous to the procedure for isolation of the homologously 
expressed avian protein. The purification protocol becomes less stringent 
as the percentage of total protein in crude extracts that is represented 
by HMG-CoA synthase increases. 
When one constructs an expression system with an HMG-CoA synthase sequence, 
regulatory sequences and suitable host, it will then be necessary to 
examine the protein preparation for specific activity, proteolytic 
cleavage, and stability after 4.degree. C. storage as described below and 
in the Examples. 
2. Analysis of Recombinant HMG-CoA Synthase 
a. Measurement of Specific Activity 
The synthase preparation of the present invention has a specific activity 
of at least 0.024 units/mg., preferably at least 0.24 units/mg. The 
Examples below disclose specific activity measurements during various 
stages of recombinant protein purification. A unit of synthase activity is 
defined as the amount of enzyme that will catalyze conversion of one 
micromole of each substrate (i.e. acetyl-CoA and acetoacetyl-CoA) to 
product HMG-CoA in one minute. 
Miziorko, et al., 1975, supra (hereby incorporated by reference) discloses 
a preferable specific activity determination. This method is described 
fully in the Examples. In brief, the time dependent conversion of [.sup.14 
C]acetyl-CoA to acid stable [.sup.14 C] HMG-CoA is measured. Alternately, 
a spectrophotometric approach can be used; this involves measurement of 
the time dependent decrease in ultraviolet absorbance due to 
acetoacetyl-CoA, as this substrate is converted (upon reaction with 
acetyl-CoA) to the product of the reaction, which is HMG-CoA. 
b. Measurement of Proteolytic Cleavage 
In a preferred form of the present invention, at least 90% of the 
recombinant synthase preparation has not been substantially 
proteolytically cleaved. By "not substantially proteolytically cleaved" we 
mean that only the initial met residue has been cleaved. 
The level of proteolytic cleavage is most preferably measured by Edman 
degradation to determine the N-terminal sequence of protein chains in the 
enzyme preparation. 
c. Measurement of Stability After 4.degree. C. Storage 
In a preferred form of the present invention, the synthase preparation is 
stable at long-term (6 month) 4.degree. C. storage. Preferably, the 
preparation retains 50% activity after one year of 4.degree. C. storage. 
The Examples below disclose preferred methods for determining enzyme 
activity. 
3. Analysis of Anti-cholesterol or Anti-Isoprene Drugs 
The present invention is also a method of analyzing candidate drugs for 
their efficacy as anti-cholesterol or anti-isoprene agents. This analysis 
involves an observation of the effect that the candidate drug has on an 
HMG-CoA synthase preparation and, therefore, a cholesterol or isoprene 
biosynthesis. A drug capable of inactivating or modifying the synthase 
preparation is a good candidate for further study as a drug useful to 
combat high cholesterol biosynthesis that could contribute to elevated 
serum cholesterol levels. 
The literature contains examples of previous attempts to study the 
interaction of various drugs with HMG-CoA synthase. For example, 
Greenspan, et al. (1993, Biochem J. 289, 889-895) discloses the inhibition 
of HMG-CoA synthase and cholesterol biosynthesis by beta-lactone 
inhibitors and the binding of these inhibitors to the enzyme. These 
workers analyzed a partially purified preparation of rat liver cytosolic 
HMG-CoA synthase with beta-lactone L-659,699 and its radioactive 
derivative. After determining that the lactone affected the synthase, the 
workers also examined cultured HepG2 cells in their ability to incorporate 
acetate into sterols. They found that sterol biosynthesis in cultured 
HepG2 cells was rapidly restored upon removal of the compound from the 
medium. 
In general, investigations such as the Greenspan, et al., 1993, supra, 
involve the exposure of an HMG-CoA synthase protein preparation to the 
candidate drug or agent and the observation of how that drug or agent 
interacts with the protein preparation. Activity measurements (such as 
those disclosed below in the Examples) can be used to assess whether or 
not the HMG-CoA synthase remains in an active state. Activity measurements 
in crude native cellular fractions may be problematic due to interference 
from other enzymes or metabolites. Straightforward estimates of whether a 
potential drug affects HMG-CoA synthase activity are facilitated by 
availability and used of isolated recombinant HMG-CoA synthase enzyme with 
increased stability. 
EXAMPLES 
The Examples below describe the isolation and characterization of HMG-CoA 
synthase expressed in E. coli. The quality of this recombinant enzyme 
preparation is evaluated by comparison of its properties with natively 
isolated avian liver enzyme. Such a comparison also demonstrated that the 
isolated avian cDNA encodes the cholesterogenic isozyme. 
2. Experimental Procedures 
a. Materials 
E. coli BL21(DE3) and pET-3d vector were purchased from Novagen (Madison, 
Wis.). E. coli DH5.alpha. were obtained from Bethesda Research Laboratory 
(Gaithersburg, Md.). Restriction enzymes, T4 DNA ligase and vent DNA 
polymerase were purchased from New England Biolabs (Beverly, Mass.). 
Sequenase and IPTG (isopropylthiogalactoside) were provided by United 
States Biochemicals (Cleveland, Ohio). 
R.(3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl;3-carboxy-PROXYL) was 
obtained from Aldrich (Milwaukee, Wis.). R.CoA thioester was synthesized 
using the mixed anhydride, prepared by activation of the free acid using 
the method of Bernert, et al., (1977) J. Biol. Chem. 252, 6736-6744. 
Deoxyoligonucleotides were synthesized by the Protein/Nucleic Acid 
Facility at the Medical College of Wisconsin. Geneclean is a product of 
Bio 101 Inc. (Vista, Calif.). All other reagents were purchased from Sigma 
Chemical Company (St. Louis, Mo.), Pharmacia (Milwaukee, Wis.) or Bio-Rad 
(Richmond, Calif.). Antiserum against rat cytosolic synthase was kindly 
provided by Dr. Michael Greenspan (Merck, Sharpe, & Dohme; Rahway, N.J.). 
b. Methods 
Construction of the Expression Vector. Isolation of full length cDNA 
encoding avian HMG-CoA synthase from a .lambda.gt11 library has been 
reported (Kattar-Cooley et al., supra, 1990). The cDNA encoding the 
synthase was derived from EcoRI insert of the .lambda. clone NC9, 
subcloned into pUC13 (pKC5). FIG. 2 describes the cloning of the synthase 
cDNA into an E. coli expression vector. The ATG codon within the Nco I 
recognition site is in frame and functions as the translation initiation 
codon, encoding a methionine as residue 1. In this expression construct, 
alanine replaces a non-conserved proline as residue 2. The open, filled, 
and stippled segments in FIG. 2 represent the coding sequence, 
untranslated region, and ampicill in resistance gene, respectively. 
Linkers are represented by striped regions. 
Referring to FIG. 2, after endonucleolytic digestion with SmaI and EcoRI, 
the 3'-terminus of SmaI-EcoRI insert was filled in with the Klenow 
fragment of DNA polymerase. Decameric linkers bearing an NcoI recognition 
sequence were ligated to the blunt-ended insert. The resulting fragment 
was purified and restricted with NcoI endonuclease to facilitate ligation 
(Maniatis et al., (1982) Molecular Cloning, Cold Spring Harbor, N.Y. pp. 
1.53-1.72) into the NcoI cloning site of the expression vector pET-3d. The 
ligation mix was used to transform DH5.alpha. competent cells (Hanahan et 
al., (1991) Methods Enzymol., 204, 63-113). Clones containing the insert 
in the desired orientation (as verified by restriction mapping) were 
isolated (Birnboim, et al., (1979) Nucleic Acid Res., 7, 1513-1523) and 
the DNA sequence encoding the N-terminus of the expression target was 
confirmed by dideoxy chain termination method (Sanger et al., (1977) Proc. 
Natl. Acad. Sci. USA 74, 5463-5467). E. coli BL21(DE3) cells were 
transformed with the purified plasmid pACS for HMG-CoA synthase 
expression. 
Bacterial Growth and Purification of HMG-CoA Synthase. A single colony of 
E. coli BL21(DE3) harboring the recombinant plasmid was grown to 
stationary phase in LB media (Miller, (1972) Experiments in Molecular 
Genetics, Cold Spring Harbor, N.Y. pp. 431-435) containing 200 .mu.g/ml 
ampicillin. The culture was diluted (1:100) with 3 liters of media of the 
same composition and grown at 30.degree. C. on a gyrotory shaker. 
Expression was induced by addition of 1 mM IPTG to culture at an O.D. of 
0.6-0.8. The cells were subsequently harvested by low speed centrifugation 
after the culture reached an O.D. of 2.2. The pellet was resuspended in 
lysis buffer (20 mM sodium phosphate pH 7.0, 1 mM EDTA, 1 mM DTT, 0.1 mM 
PMSF, 10% glycerol) and lysed in a French pressure cell at 16,000 psi. 
Supernatant was recovered from the crude extract by centrifugation at 
46,000.times.g for 45 minutes. 
HMG-CoA synthase was purified from the supernatant by an adaptation of the 
procedure of Clinkenbeard et al., (supra). An ammonium sulfate fraction 
(30-45% saturation), prepared from the supernatant, was dissolved in 20 mM 
sodium phosphate buffer pH 6.5, 0.1 mM EDTA, 0.1 mM DTT and dialyzed 
overnight against 20 liters of buffer of the same composition. The 
dialysate was loaded onto a 2.5.times.64 cm DEAE-cellulose column 
equilibrated with 20 mM sodium phosphate buffer, pH 6.5, containing 0.1 mM 
EDTA, and 0.1 mM DTT. The column was washed with 2 column volumes of the 
equilibration buffer and eluted using 1.6 liters of a 20-160 mM linear 
gradient of sodium phosphate buffer, pH 6.5, containing 0.1 mM EDTA and 
0.1 mM DTT. The fractions containing HMG-CoA synthase activity were pooled 
and concentrated using an Amicon ultrafiltration cell. 100-150 mg of 
enzyme was isolated from a 3 liter culture. 
Western Blotting. Methodology reported by Haas, et al., (1985, J. Biol. 
Chem., 260, 12464-12473), was employed for immunochemical detection of 
HMG-CoA synthase. Crude E. coli extract was electrophoresed on an 
SDS-polyacrylamide gel. The proteins were transferred to a nitrocellulose 
membrane, followed by incubation with a 1:1000 dilution of antiserum 
raised against avian mitochondrial synthase or rat cytosolic synthase and 
subsequent washing to eliminate non-specific binding. The resulting 
complex was visualized by incubation with a solution of [.sup.125 I] 
protein A, followed by autoradiography. 
Characterization of the Recombinant Enzyme. For measurement of the overall 
condensation reaction (Miziorko et al., (1975) J. Biol. Chem. 250, 
5678-5773), 200 .mu.M acetyl-CoA was added to a reaction mixture 
(30.degree. C.) containing 100 mM Tris-HCl pH 8.2, 100 .mu.M EDTA, 50 
.mu.M acetoacetyl-CoA and appropriately diluted HMG-CoA synthase 
(approximately 6 .mu.g wild type enzyme in 1.0 ml final volume). The 
reaction rate was monitored by acetyl-CoA dependant loss of absorbance at 
300 nm, due to condensation with the enolate of acetoacetyl-CoA. 
The enzyme's specific activity was calculated as .mu.moles/min/mg. (This is 
equivalent to units/mg, since 1 unit=1 umole/min.) Apparent K.sub.m 
measurement was done in the presence of 20 .mu.M acetoacetylCoA. The 
enzyme's acetyl-CoA hydrolase activity was determined (Miziorko et al., 
1975, supra) by measuring the time dependent depletion of [.sup.14 
C]acetyl-CoA, after its conversion to acid stable citrate upon reaction 
with excess citrate synthase and oxaloacetate. The reaction mixture 
contained 100 mM potassium phosphate, pH 8.0, and 60 .mu.g HMG-CoA 
synthase in 300 .mu.l total volume at 30.degree. C. The reaction was 
initiated by addition of 100 .mu.M [.sup.14 C] acetyl-CoA (10,000 
dpm/nmole). At specific time intervals, 20 .mu.l aliquots were removed and 
added rapidly to a mixture containing 500 milliunits of citrate synthase 
and 400 .mu.M oxaloacetate in 100 mM potassium phosphate buffer, pH 8.0 
(100 .mu.l final volume). The resulting mix was acidified with 100 .mu.l 
of 6N HCl and heated to dryness. The acid stable radioactivity which is 
measured is due to unhydrolyzed [.sup.14 C] acetyl-CoA. 
The stoichiometry of acetyl-CoA binding was determined according to the 
procedure of Vollmer et al. (1988, supra). After 30.degree. C. incubation 
of the enzyme (120 .mu.g) in 100 mM sodium phosphate, pH 7.5, the mixture 
was placed on ice. [.sup.14 C]acetyl-CoA (10,000 dpm/nmole) was added to 
bring the 100 .mu.l incubation mixture to a final concentration of 200 
.mu.M. Unbound acetyl CoA was removed using a G-50 centrifugal column 
equilibrated with 10 mM sodium acetate, pH 5.0, at 4.degree. C. Protein in 
the recovered samples was estimated by the Bradford assay ((1976) Anal. 
Biochem., 72, 248-254) and radioactivity was determined by liquid 
scintillation counting. 
Stoichiometry of covalent acetylation was determined according to Miziorko 
et al. (1975, supra). The incubation mixture, containing [.sup.14 
C]acetyl-CoA and 50 .mu.g wild type or 300 .mu.g mutant enzyme in 100 
.mu.l, was treated with 900 .mu.l of ice-cold 10% trichloroacetic acid. 
The denatured protein was recovered by centrifugation. The pellet was 
resuspended in 10% trichloroacetic acid and transferred to a glass fiber 
filter. The filters were washed extensively with ice-cold 10% 
trichloroacetic acid and 50 mM sodium pyrophosphate in 500 mM HCl, and 
once with cold absolute ethanol. Filters were dried and radioactivity was 
determined by liquid scintillation counting. 
Measurement of R.CoA Binding by EPR. Conventional X-band EPR spectra were 
recorded using a Varian Century-Line 9-GHz spectrometer. The samples 
contained variable concentrations of HMG-CoA synthase sites (0-930 .mu.M) 
in 50 mM sodium phosphate buffer, pH 7.0, and 50 or 150 .mu.M R.CoA. R.CoA 
bound to HMG-CoA synthase was calculated by comparing the amplitudes of 
high, center, or low-field lines of sample spectra to the corresponding 
lines observed with a solution containing an equal concentration of R.CoA 
in buffer. The data were analyzed (Miziorko et al., (1979) Biochemistry 
18, 399-403) by Scatchard plot using linear regression analysis. The 
spectra were recorded at ambient temperature with a modulation amplitude 
of 1 G, modulation frequency of 100 KHz and microwave power 5 mW. Field 
sweep was 100 G and time constant was 0.5 sec. The EPR spectrum of bound 
R.CoA was obtained at 5 G modulation amplitude and variable gain. 
Rotational correlation time of the bound spin label was determined using a 
spectral simulation algorithm (Freed, (1976) Spin Labeling: Theory and 
Applications (L. Berliner, ed.) 1, 53-132; Schneider, et al. (1989) 
Biological Magnetic Resonance 8, 1-76). 
Measurement of Proteolytic Cleavage. Evaluation of the degree of 
proteolysis of the recombinant HMG-CoA synthase was based on N-terminal 
sequence analysis by the Edman degradation procedure. Typically &gt;100 
picomoles of protein are used for analysis, which was carried out using an 
Applied Biosystems model 477A pulsed liquid phase sequencer equipped with 
online PTH analyzer (Model 120) to detect the phenylthiohydatoin 
derivatives of amino acids released at each step in the analysis. Ten 
Edman cycles were usually sufficient to characterize the preparation. 
3. Results 
Expression and Isolation of Recombinant HMG-CoA Synthase. T7 
polymerase-dependent protein synthesis was induced by addition of IPTG to 
the E. coli BL21(DE3) culture that has been transformed with expression 
plasmid pACS, which contains synthase-encoding cDNA. FIG. 3, a 
Coomassie-stained SDS-polyacrylamide gel, is a demonstration of the 
expression of cytosolic HMG-CoA synthase in E. coli. Lane 1 is the avian 
liver mitochondrial HMG-CoA synthase (2 micrograms); lanes 2, 3 and 4 are 
total extract, 46,000.times.g supernatant, and 10,000.times.g supernatant, 
respectively from E. coli harboring the expression plasmid (12 microgram 
protein); lanes 5, 6 and 7 are fractions equivalent to those described for 
lanes 2, 3 and 4, but are derived from bacterial cells carrying the vector 
plasmid only (12 micrograms protein). There was a marked accumulation of 
protein (FIG. 3, lane 2) which exhibits a subunit molecular weight in 
excess of that observed for a liver mitochondrial synthase marker (FIG. 3, 
lane 1). No comparable protein appeared upon addition of IPTG to bacteria 
containing only the parent pET-3d vector (FIG. 3, lanes 5-7). There is a 5 
kDa increment in subunit molecular weight for the avian cytosolic HMG-CoA 
synthase (Clinkenbeard et al., 1975, supra) over that of the mature 
mitochondrial isozyme (Reed et al., (1975) J. Biol. Chem. 250, 3117-3123). 
Appearance of this prominent Coomassie-stained band prompted activity 
assays which demonstrated high levels of HMG-CoA synthase activity in 
extracts of bacteria that harbor the expression plasmid and no activity in 
samples from bacteria containing the pET-3d vector. Table 1, below, 
summarizes these results. Based on the specific activity of purified 
synthase (vide infra), approximately 24% of protein in the crude bacterial 
extracts was attributable to the target of T7 polymerase dependent 
expression. Importantly, virtually all of the expressed protein is active 
and soluble, as judged from measurements on high speed supernatants (FIG. 
3, lanes 3,4; Table 1). 
TABLE 1 
______________________________________ 
Total Specific 
Purification 
Protein Total Activity 
Purification 
Yield 
step (mg) units (Units/mg) 
(Fold) (%) 
______________________________________ 
Crude Extract 
1,820 451 0.24 (1) (100) 
High Speed 1,732 407 0.23 0.96 90 
Supernatant 
30-45% (NH.sub.4).sub.2 SO.sub.4 
623 302 0.48 2.0 67 
Fractionation 
DEAE-Cellulose 
150 151 1.00 4.1 33 
Chromatography 
______________________________________ 
The level of overexpression facilitated isolation of significant amounts of 
homogeneous enzyme by application of rudimentary salt fractionation and 
ion exchange chromatography procedures (FIG. 4; Table I). FIG. 4 is an SDS 
polyacrylamide electrophoretic gel containing recombinant HMG-CoA 
synthase. The samples correspond to the recombinant wild-type enzyme at 
various stage of purification (Lanes 3-6) and a modified recombinant HMG 
synthase in Lane 7. Lane 1 represents molecular weight markers (serum 
albumin, 66.2 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; soybean 
trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa). Lane 2 is mitochondrial 
HMG-CoA synthase isolated from chicken liver. Lane 3 is total extract of 
E. coli containing the plasmid encoding wild-type HMG-CoA synthase (12 
micrograms). Lane 4 is a supernatant (10 micrograms) obtained after 
centrifugation of bacterial extract at 46,000.times.g for 45 minutes. Lane 
5 is the 30-45% (NH.sub.4).sub.2 SO.sub.4 fraction (10 micrograms). Lane 6 
is a DEAE eluate (10 micrograms). Lane 7 is a purified mutated recombinant 
HMG-CoA synthase. 
Specific activity of the isolated recombinant synthase is in excellent 
agreement with the highest values reported for the avian liver cytosolic 
enzyme (Clinkenbeard et al., 1975, supra). Unlike that enzyme, which was 
vulnerable to proteolysis, expression of our construct in E. coli results 
in an enzyme that is quite stable in crude extracts, during isolation, and 
upon long term storage. We determined that the preparation in its final 
purified form had 50% activity after one year at 4.degree. C. storage and 
negligible loss at -80.degree. C. storage. 
Properties of Recombinant HMG-CoA Synthase. Table 2, below, is a comparison 
of the characteristics of our recombinant HMG-CoA synthase and native 
avian liver HMG-CoA synthases. In addition to comparable catalytic 
activity in the overall condensation reaction to form HMG-CoA, the 
apparent Michaelis constant was in excellent agreement with that estimated 
for the liver enzyme. The stoichiometry with which the covalent the 
acetyl-S-enzyme intermediate was trapped on the recombinant enzyme (Table 
2) was also in good agreement with the range of values observed using 
various avian liver preparations. In the absence of the second substrate, 
acetoacetyl-CoA, HMG-CoA synthase catalyzes hydrolysis of acetyl-CoA 
(Miziorko et al., 1975, supra). The recombinant enzyme also catalyzes this 
partial reaction, exhibiting a rate and Michaelis constant (Table 2) that 
are in good agreement with that earlier report. It had been previously 
established that avian HMG-CoA synthase specifically binds a spin-labeled 
substrate analog, R.CoA, as a competitive inhibitor with respect to 
acetyl-CoA (Miziorko et al., 1979, supra). When evaluated for interaction 
with recombinant enzyme, R.CoA was found to bind at a stoichiometry of 
0.9/protomer and with a K.sub.d =102 uM, in agreement with the values 
reported for the avian enzyme. Moreover, simulation of the spectral 
features of the immobilized spin-labeled acyl-CoA (Freed, 1976, supra; 
Schneider, et al., 1989, supra) suggested a rotational correlation time, 
.tau..sub.c =35 nanoseconds, which agrees with the range of values 
estimated for the avian enzyme. This .tau..sub.c value for the bound 
spin-probe predicts, according to the Stokes-Einstein equation, virtually 
complete immobilization of the acyl group on a 115 Kda protein, in 
agreement with the dimeric nature of the native, avian enzyme. 
TABLE 2 
______________________________________ 
Parameters Liver Enzyme.sup.a 
Recombinant Enzyme.sup.b 
______________________________________ 
Specific Activity 
0.5-1.0 0.8-1.0 
(.mu.mol/min/mg) 
K.sub.m AcCoA (Overall Rxn.; .mu.M) 
300 270 
Acetylation stoichiometry 
0.50-0.75 0.62 
(mol/mol subunit) 
Effect of MgCl.sub.2 on activity 
40% increase 
45% increase 
Hydrolase Activity 
1.0 .times. 10.sup.-2 
1.8 .times. 10.sup.-2 
(.mu.mol/min/mg) 
K.sub.m AcCoA (Hydrolase Rxn.; uM) 
14 12 
______________________________________ 
In the overall condensation reaction, K.sub.m is an apparent value, as th 
conventional assay is performed in presence of 20 .mu.M acetoacetylCoA, 
which is slightly inhibitory. 
.sup.a Data for the liver enzyme are taken from Clinkenbeard et al (1975) 
and Miziorko et al (1975). 
.sup.b Data for recombinant synthase are from this report, employing 
methodology described in detail in experimental procedures. 
Upon Edman degradation analysis (10 cycles) of the purified recombinant 
HMG-CoA synthase, we determined that 90% of protein chains lacked the 
N-terminal methionine that was encoded by the AUG start codon in the cDNA. 
This processing of the N-terminal methionine is commonplace in bacteria. 
Thus, these proteins have an N-terminal alanine, which was encoded by the 
second codon of the modified cDNA as engineered and ligated into pACS. The 
remaining 10% of the preparation can largely be attributed to protein 
chains from which the N-terminal methionine has not been processed. No 
indication of extensive proteolysis during isolation from bacterial 
extract of the recombinant HMG-CoA synthase was apparent by the stringent 
criterion represented in these N-terminal analyses. 
Recombinant HMG-CoA Synthase Represents the Cholesterogenic Isozyme. The 
primary sequence of the protein, as deduced from avian cDNA sequence data, 
has been assigned to the cytosolic, cholesterogenic HMG-CoA synthase 
(Kattar-Cooley et al., 1990, supra). This evaluation was based on 
comparison between the deduced sequence and the empirically determined 
(Edman degradation) sequence of a series of peptides isolated from the 
mitochondrial, ketogenic enzyme. Availability of the isolated recombinant 
enzyme facilitates further tests of this assignment. In addition to the 
observation of the predicted increment in subunit molecular weight that 
distinguishes mitochondrial and recombinant enzymes (FIG. 4), further 
differences were apparent upon immunochemical analysis. 
FIG. 5 is a set of immunoblots prepared by reaction of HMG-CoA synthase 
samples with antiserum raised against avian mitochondrial HMG-CoA synthase 
(A) or rat cytosolic HMG-CoA synthase (B). Lane 1 is avian liver 
mitochondrial HMG-CoA synthase (0.2 micrograms). Lane 2 is 2.5 micrograms 
of crude extract of IPTG-induced E. coli harboring the expression plasmid 
pACS. Antigen-antibody complexes are detected by autoradiography after 
reaction with [.sup.125 I] protein A. Panel B is intentionally overexposed 
to allow visualization of the weak cross-reaction between avian 
mitochondrial antigen and antiserum prepared against rat cytosolic 
antigen. 
Referring to FIG. 5, in western blot experiments, antiserum prepared 
against isolated avian mitochondrial enzyme (Miziorko, 1985, supra) 
sensitively detected this antigen (FIG. 4a) as well as the recombinant 
synthase. However, when antiserum prepared against the rodent cytosolic, 
cholesterogenic synthase (Mehrabian et al., (1986) J. Biol. Chem. 261, 
16249-16255) was tested using an identical blot of the avian cDNA-encoded 
proteins, it detected the recombinant synthase with much higher 
sensitivity than the avian mitochondrial protein (FIG. 5b). In retrospect, 
this discrimination, which was consistent with the assignment of the 
recombinant enzyme as the cytosolic isozyme is quite reasonable. The 
homology between cytosolic synthases from different eukaryotes (rat vs. 
chicken, 84% identity) is considerably higher than the homology between 
cytosolic and mitochondrial proteins from the same species (65% identity 
for the rat enzymes; Ayte et al., (1990) Proc. Natl. Acad. Sci. USA 87, 
3874-3878; Casals et al., 1992, supra). The recombinant enzyme displays a 
1.4 fold stimulation of catalytic activity in the presence of Mg.sup.2+ 
(Table 2). This behavior, previously reported for the avian cytosolic 
enzyme (Clinkenbeard et al., 1975, supra), distinguishes it from the 
mitochondrial isozyme, which is inhibited by Mg.sup.2+ (Reed et al., 1975, 
supra). Finally, the empirically determined pI of recombinant synthase 
(5.8; minor bands at 5.6, 5.4) agreed well with our calculated estimate of 
pI=5.6 (Kattar-Cooley et al., 1990, supra) as well as empirical estimates 
for avian (5.2; 5.4; 6.6) and rat (5.4) cytosolic synthases (Clinkenbeard 
et al., 1975, supra), further distinguishing this protein from the more 
basic (pI=7.2; Reed et al., 1975, supra) mitochondrial isozyme. Minor 
proteolysis may account for microheterogeneity apparent in pI 
determinations. 
4. Discussion 
In developing a strategy for expression of a recombinant form of avian 
HMG-CoA synthase, advantage was taken of the fact that the ATG start codon 
represents half of the Nco I recognition sequence. Modification of the 
original cDNA to encode a complete Nco I site resulted in a second codon 
that translates into alanine instead of proline at residue 2. The mature 
form of the avian mitochondrial enzyme does not contain a corresponding 
sequence (Kattar-Cooley et al., 1990, supra), yet it functions with 
catalytic efficiency that is comparable to the cytosolic enzyme. In a 
sequence deduced from cDNA proposed to encode the precursor form of rat 
mitochondrial synthase (Ayte et al, 1990, supra), the N-terminus of the 
mature protein was not directly identified but a conserved proline did not 
appear in the vicinity of the region generally expected to represent the 
N-terminus of the processed matrix protein. The validation of this 
strategy for expression of functional synthase is apparent upon comparison 
of the properties of isolated avian liver and recombinant enzymes, which 
is facilitated by the high level of expression and stability of the latter 
protein. Regardless of whether catalytic efficiency, substrate binding, or 
Mg.sup.2+ stimulation are considered, recombinant enzyme faithfully 
reflects the homologously expressed avian protein. 
The partial reactions catalyzed by HMG-CoA synthase are valuable tools for 
characterizing the recombinant enzyme, regardless of whether it represents 
wild type protein or an engineered variant. In the absence of 
co-substrate, HMG-CoA synthase hydrolyzes acetyl-CoA (Miziorko et al., 
1975, supra). In catalyzing this partial reaction, it is similar to 
chloramphenicol acetyltransferase, although that protein's relative rate 
of hydrolysis (0.1% of overall reaction; Kleanthous, et al., (1984) 
Biochem. J. 223, 211-220) is lower than the 1% relative hydrolysis rate 
reported for HMG-CoA synthase (Miziorko et al., 1975, supra). Such 
hydrolysis is not unexpected, given the thioesterase activity inherent in 
synthase (cf. eqn.3). The excellent agreement not only between relative 
rates of hydrolysis catalyzed by avian and recombinant synthases (Table 2) 
but also between their respective K.sub.mAc-CoA 's in this partial 
reaction certainly supports the use of recombinant protein as an 
experimental model. Additional support derives from the enzymes' 
acetylation partial reaction (eqn. 1); covalently bound acetyl groups can 
be trapped with good stoichiometry on both homologously and heterologously 
expressed synthases. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1824 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CATGGCTGGGTCTCTTCCAGTGAACACTGAATCCTGCTGGCCCAAAGATGTGGGTATTGT60 
TGCACTGGAAATCTATTTTCCCTCTCAGTATGTCGACCAGACTGAGCTGGAGAAGTATGA120 
CGGTGTGGATGCAGGCAAATACACCATTGGGTTAGGCCAGTCAAAGATGGGCTTCTGCTC180 
TGACCGAGAGGATATCAATTCCCTCTGTTTGACTGTCGTTCAGAAGCTTATGGAGAGGAA240 
CAGCCTTTCCTATGACTGCATTGGGAGACTGGAAGTTGGAACGGAGACAATAATTGATAA300 
ATCAAAATCGGTGAAGACTGTCCTGATGCAGCTATTTGAAGAATCTGGTAATACAGATGT360 
AGAAGGAATTGACACAACCAATGCGTGCTATGGAGGCACTGCTGCTCTTTTTAATGCTAT420 
TAACTGGATTGAGTCCAGTTCTTGGGATGGACGCTATGCACTTGTTGTTGCTGGAGACAT480 
TGCTGTGTATGCCACTGGAAATGCCAGGCCAACAGGTGGAGCTGGTGCTGTTGCTATGCT540 
AGTTGGGTCAAATGCTCCTTTAATTTTTGAGAGAGGATTGCGTGGAACCCACATGCAGCA600 
TGCTTATGACTTCTATAAACCAGATATGGTTTCTGAATATCCTGTAGTTGATGGCAAACT660 
ATCTATACAGTGCTACCTCAGTGCATTAGACCGCTGCTATAGTGTTTATCGCAATAAAAT720 
CCATGCCCAGTGGCAAAAAGAGGGGACAGACAGAGGTTTCACCTTGAATGATTTTGGATT780 
CATGATCTTTCATTCTCCCTACTGTAAACTGGTACAGAAGTCGGTGGCAAGACTGTTGCT840 
GAATGACTTTCTCAGTGACCAGAATGCAGAAACAGCAAATGGTGTTTTCAGTGGTCTGGA900 
AGCTTTCAGGGATGTAAAGCTTGAAGATACATATTTTGATAGGGATGTGGAAAAAGCTTT960 
TATGAAAGCTAGTGCAGAGCTCTTCAATCAGAAAACCAAAGCTTCCTTACTTGTGTCCAA1020 
TCAGAATGGAAACATGTACACGCCTTCAGTCTACGGTTGCCTTGCTTCTCTTCTAGCCCA1080 
GTACTCTCCAGAGCACCTTGCAGGACAAAGAATCAGTGAGTTCTCATATGGCTCTGGTTT1140 
TGCTGCTACGCTGTATTCCATCAGAGTTACACAGGATGCCACTCCTGGTTCTGCGCTTGA1200 
CAAAATAACTGCTAGCCTTTCTGATCTTAAAGCAAGACTTGACTCACGAAAATGCATTGC1260 
ACCTGATGTCTTTGCTGAAAACATGAAGATTAGACAGGAGACACATCACTTGGCCAACTA1320 
TATTCCACAGTGTTCAGTAGAAGATCTCTTTGAGGGAACATGGTATCTTGTGCGTGTGGA1380 
TGAAAAACACAGGAGAACATATGCACGACGCCCAGTTATGGGTGATGGACCCCTGGAGGC1440 
AGGAGTTGAAGTTGTCCACCCAGGCATTGTTCATGAGCACATCCCAAGCCCTGCTAAGAA1500 
AGTGCCAAGAATCCCTGCAACAACAGAATCTGAAGGCGTTACTGTTGCCATTTCCAATGG1560 
GGTGCATTAAGATACCTCTGTGAGGCAAGAAAGAAGAAACTGGCCTTATGAAAACTTGAA1620 
GTTCTGGGGTCACAGTCTTTGTGGACCTCCCGGATCGTTCTCTTTAATATTTCATTACAA1680 
AAAAATGGAAAGAAAGACTTGCTGGGGCTTGTGGAAAATCATTATTTCAAGGCTTTGTTT1740 
CAAGATAACTTAAATGTTTTCAGGTGGCATCTGTTCATAGTGGAGAAATTCATTCACAGC1800 
CTGTCTCAAATTAACTGAATTAGC1824 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 522 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetProGlySerLeuProValAsnThrGluSerCysTrpProLysAsp 
151015 
ValGlyIleValAlaLeuGluIleTyrPheProSerGlnTyrValAsp 
202530 
GlnThrGluLeuGluLysTyrAspGlyValAspAlaGlyLysTyrThr 
354045 
IleGlyLeuGlyGlnSerLysMetGlyPheCysSerAspArgGluAsp 
505560 
IleAsnSerLeuCysLeuThrValValGlnLysLeuMetGluArgAsn 
65707580 
SerLeuSerTyrAspCysIleGlyArgLeuGluValGlyThrGluThr 
859095 
IleIleAspLysSerLysSerValLysThrValLeuMetGlnLeuPhe 
100105110 
GluGluSerGlyAsnThrAspValGluGlyIleAspThrThrAsnAla 
115120125 
CysTyrGlyGlyThrAlaAlaLeuPheAsnAlaIleAsnTrpIleGlu 
130135140 
SerSerSerTrpAspGlyArgTyrAlaLeuValValAlaGlyAspIle 
145150155160 
AlaValTyrAlaThrGlyAsnAlaArgProThrGlyGlyAlaGlyAla 
165170175 
ValAlaMetLeuValGlySerAsnAlaProLeuIlePheGluArgGly 
180185190 
LeuArgGlyThrHisMetGlnHisAlaTyrAspPheTyrLysProAsp 
195200205 
MetValSerGluTyrProValValAspGlyLysLeuSerIleGlnCys 
210215220 
TyrLeuSerAlaLeuAspArgCysTyrSerValTyrArgAsnLysIle 
225230235240 
HisAlaGlnTrpGlnLysGluGlyThrAspArgGlyPheThrLeuAsn 
245250255 
AspPheGlyPheMetIlePheHisSerProTyrCysLysLeuValGln 
260265270 
LysSerValAlaArgLeuLeuLeuAsnAspPheLeuSerAspGlnAsn 
275280285 
AlaGluThrAlaAsnGlyValPheSerGlyLeuGluAlaPheArgAsp 
290295300 
ValLysLeuGluAspThrTyrPheAspArgAspValGluLysAlaPhe 
305310315320 
MetLysAlaSerAlaGluLeuPheAsnGlnLysThrLysAlaSerLeu 
325330335 
LeuValSerAsnGlnAsnGlyAsnMetTyrThrProSerValTyrGly 
340345350 
CysLeuAlaSerLeuLeuAlaGlnTyrSerProGluHisLeuAlaGly 
355360365 
GlnArgIleSerGluPheSerTyrGlySerGlyPheAlaAlaThrLeu 
370375380 
TyrSerIleArgValThrGlnAspAlaThrProGlySerAlaLeuAsp 
385390395400 
LysIleThrAlaSerLeuSerAspLeuLysAlaArgLeuAspSerArg 
405410415 
LysCysIleAlaProAspValPheAlaGluAsnMetLysIleArgGln 
420425430 
GluThrHisHisLeuAlaAsnTyrIleProGlnCysSerValGluAsp 
435440445 
LeuPheGluGlyThrTrpTyrLeuValArgValAspGluLysHisArg 
450455460 
ArgThrTyrAlaArgArgProValMetGlyAspGlyProLeuGluAla 
465470475480 
GlyValGluValValHisProGlyIleValHisGluHisIleProSer 
485490495 
ProAlaLysLysValProArgIleProAlaThrThrGluSerGluGly 
500505510 
ValThrValAlaIleSerAsnGlyValHis 
515520 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 507 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetGlnArgLeuLeuAlaProAlaArgArgValLeuGlnValLysArg 
151015 
ValMetGlnGluSerSerLeuSerProAlaHisLeuLeuProAlaAla 
202530 
GlnGlnArgPheSerThrIleProProAlaProLeuAlaLysThrAsp 
354045 
ThrTrpProLysAspValGlyIleLeuAlaLeuGluValTyrPhePro 
505560 
AlaGlnTyrValAspGlnThrAspLeuGluLysPheAsnAsnValGlu 
65707580 
AlaGlyLysTyrThrValGlyLeuGlyGlnThrArgMetGlyPheCys 
859095 
SerValGlnGluAspIleAsnSerLeuCysLeuThrValValGlnArg 
100105110 
LeuMetGluArgThrLysLeuProTrpAspAlaValGlyArgLeuGlu 
115120125 
ValGlyThrGluThrIleIleAspLysSerLysAlaValLysThrVal 
130135140 
LeuMetGluLeuPheGlnAspSerGlyAsnThrAspIleGluGlyIle 
145150155160 
AspThrThrAsnAlaCysTyrGlyGlyThrAlaSerLeuPheAsnAla 
165170175 
AlaAsnTrpMetGluSerSerTyrTrpAspGlyArgTyrAlaLeuVal 
180185190 
ValCysGlyAspIleAlaValTyrProSerGlyAsnProArgProThr 
195200205 
GlyGlyAlaGlyAlaValAlaMetLeuIleGlyProLysAlaProLeu 
210215220 
ValLeuGluGlnGlyLeuArgGlyThrHisMetGluAsnAlaTyrAsp 
225230235240 
PheTyrLysProAsnLeuAlaSerGluTyrProLeuValAspGlyLys 
245250255 
LeuSerIleGlnCysTyrLeuArgAlaLeuAspArgCysTyrAlaAla 
260265270 
TyrArgArgLysIleGlnAsnGlnTrpLysGlnAlaGlyAsnAsnGln 
275280285 
ProPheThrLeuAspAspValGlnTyrMetIlePheHisThrProPhe 
290295300 
CysLysMetValGlnLysSerLeuAlaArgLeuMetPheAsnAspPhe 
305310315320 
LeuSerSerSerSerAspLysGlnAsnAsnLeuTyrLysGlyLeuGlu 
325330335 
AlaPheLysGlyLeuLysLeuGluGluThrTyrThrAsnLysAspVal 
340345350 
AspLysAlaLeuLeuLysAlaSerLeuAspMetPheAsnLysLysThr 
355360365 
LysAlaSerLeuTyrLeuSerThrAsnAsnGlyAsnMetTyrThrSer 
370375380 
SerLeuGlyCysLeuAlaSerLeuLeuSerHisHisSerAlaGlnGlu 
385390395400 
LeuAlaGlySerArgIleGlyAlaPheSerTyrGlySerGlyLeuAla 
405410415 
AlaSerPhePheSerPheArgValSerLysAspAlaSerProGlySer 
420425430 
ProLeuGluLysLeuValSerSerValSerAspLeuProLysArgLeu 
435440445 
AspSerArgArgArgMetSerProGluGluPheThrGluIleMetAsn 
450455460 
GlnArgGluGlnPheTyrHisLysValAsnPheSerProProGlyAsp 
465470475480 
ThrSerAsnLeuPheProGlyThrTrpTyrLeuGluArgValAspGlu 
485490495 
MetHisArgArgLysTyrAlaArgArgProVal 
500505 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 520 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetProGlySerLeuProLeuAsnAlaGluAlaCysTrpProLysAsp 
151015 
ValGlyIleValAlaLeuGluIleTyrPheProSerGlnTyrValAsp 
202530 
GlnAlaGluLeuGluLysTyrAspGlyValAspAlaGlyLysTyrThr 
354045 
IleGlyLeuGlyGlnAlaArgMetGlyPheCysThrAspArgGluAsp 
505560 
IleAsnSerLeuCysLeuThrValValGlnAsnLeuMetGluArgAsn 
65707580 
SerLeuSerTyrAspCysIleGlyArgLeuGluValGlyThrGluThr 
859095 
IleIleAspLysSerLysSerValLysSerAsnLeuMetGlnLeuPhe 
100105110 
GluGluSerGlyAsnThrAspIleGluGlyIleAspThrThrAsnAla 
115120125 
CysTyrGlyGlyThrAlaAlaValPheAsnAlaValAsnTrpIleGlu 
130135140 
SerSerSerTrpAspGlyArgTyrAlaLeuValValAlaGlyAspIle 
145150155160 
AlaIleTyrAlaThrGlyAsnAlaArgProThrGlyGlyValGlyAla 
165170175 
ValAlaLeuLeuIleGlyProAsnAlaProLeuIlePheAspArgGly 
180185190 
LeuArgGlyThrHisMetGlnHisAlaTyrAspPheTyrLysProAsp 
195200205 
MetLeuSerGluTyrProIleValAspGlyLysLeuSerIleGlnCys 
210215220 
TyrLeuSerAlaLeuAspArgCysTyrSerValTyrArgLysLysIle 
225230235240 
ArgAlaGlnTrpGlnLysGluGlyAsnAspAsnAspPheThrLeuAsn 
245250255 
AspPheGlyPheMetIleSerHisSerProTyrCysLysLeuValGln 
260265270 
LysSerLeuAlaArgMetPheLeuAsnAspPheLeuAsnAspGlnAsn 
275280285 
ArgAspLysAsnSerIleTyrSerGlyLeuGluAlaPheGlyAspVal 
290295300 
LysLeuGluAspThrTyrPheAspArgAspValGluLysAlaPheMet 
305310315320 
LysAlaSerSerGluLeuPheAsnGlnLysThrLysAlaSerLeuLeu 
325330335 
ValSerAsnGlnAsnGlyAsnMetTyrThrSerSerValTyrGlySer 
340345350 
LeuAlaSerValLeuAlaGlnTyrSerProGlnGlnLeuAlaGlyLys 
355360365 
ArgIleGlyValPheSerTyrGlySerGlyLeuAlaAlaThrLeuTyr 
370375380 
SerLeuLysValThrGlnAspAlaThrProGlySerAlaLeuAspLys 
385390395400 
ValThrAlaSerLeuCysAspLeuLysSerArgLeuAspSerArgThr 
405410415 
CysValAlaProAspValPheAlaGluAsnMetLysLeuArgGluAsp 
420425430 
ThrHisHisLeuAlaAsnTyrIleProGlnCysSerIleAspSerLeu 
435440445 
PheGluGlyThrTrpTyrLeuValArgValAspGluLysHisArgArg 
450455460 
ThrTyrAlaArgArgProSerThrAsnAspHisAsnLeuGlyAspGly 
465470475480 
ValGlyLeuValHisSerAsnThrAlaThrGluHisIleProSerPro 
485490495 
AlaLysLysValProArgLeuProAlaThrAlaAlaGluSerGluSer 
500505510 
AlaValIleSerAsnGlyGluHis 
515520 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 520 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
MetProGlySerLeuProLeuAsnAlaGluAlaCysTrpProLysAsp 
151015 
ValGlyIleValAlaLeuGluIleTyrPheProSerGlnTyrValAsp 
202530 
GlnAlaGluLeuGluLysTyrAspGlyValAspAlaGlyLysTyrThr 
354045 
IleGlyLeuGlyGlnAlaArgMetGlyPheCysThrAspArgGluAsp 
505560 
IleAsnSerLeuCysLeuThrValValGlnLysLeuMetGluArgAsn 
65707580 
SerLeuSerTyrAspCysIleGlyArgLeuGluValGlyThrGluThr 
859095 
IleIleAspLysSerLysSerValLysSerAsnLeuMetGlnLeuPhe 
100105110 
GluGluSerGlyAsnThrAspIleGluGlyIleAspThrThrAsnAla 
115120125 
CysTyrGlyGlyThrAlaAlaValPheAsnAlaValAsnTrpIleGlu 
130135140 
SerSerSerTrpAspGlyArgTyrAlaLeuValValAlaGlyAspIle 
145150155160 
AlaIleTyrAlaSerGlyAsnAlaArgProThrGlyGlyValGlyAla 
165170175 
ValAlaLeuLeuIleGlyProAsnAlaProValIlePheAspArgGly 
180185190 
LeuArgGlyThrHisMetGlnHisAlaTyrAspPheTyrLysProAsp 
195200205 
MetLeuSerGluTyrProValValAspGlyLysLeuSerIleGlnCys 
210215220 
TyrLeuSerAlaLeuAspArgCysTyrSerValTyrArgLysLysIle 
225230235240 
ArgAlaGlnTrpGlnLysGluGlyLysAspLysAspPheThrLeuAsn 
245250255 
AspPheGlyPheMetIlePheHisSerProTyrCysLysLeuValGln 
260265270 
LysSerLeuAlaArgMetPheLeuAsnAspPheLeuAsnAspGlnAsn 
275280285 
ArgAspLysAsnSerIleTyrSerGlyLeuGluAlaPheGlyAspVal 
290295300 
LysLeuGluAspThrTyrPheAspArgAspValGluLysAlaPheMet 
305310315320 
LysAlaSerAlaGluLeuPheAsnGlnLysThrLysAlaSerLeuLeu 
325330335 
ValSerAsnGlnAsnGlyAsnMetTyrThrSerSerValTyrGlySer 
340345350 
LeuAlaSerValLeuAlaGlnTyrSerProGlnGlnLeuAlaGlyLys 
355360365 
ArgIleGlyValPheSerTyrGlySerGlyLeuAlaAlaThrLeuTyr 
370375380 
SerLeuLysValThrGlnAspAlaThrProGlySerAlaLeuAspLys 
385390395400 
IleThrAlaSerLeuCysAspLeuLysSerArgLeuAspSerArgThr 
405410415 
CysValAlaProAspValPheAlaGluAsnMetLysLeuArgGluAsp 
420425430 
ThrHisHisLeuAlaAsnTyrIleProGlnCysSerIleAspSerLeu 
435440445 
PheGluGlyThrTrpTyrLeuValArgValAspGluLysHisArgArg 
450455460 
ThrTyrAlaArgArgProSerThrAsnAspHisSerLeuAspGluGly 
465470475480 
ValGlyLeuValHisSerAsnThrAlaThrGluHisIleProSerPro 
485490495 
AlaLysLysValProArgLeuProAlaThrSerGlyGluProGluSer 
500505510 
AlaValIleSerAsnGlyGluHis 
515520 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 520 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetProGlySerLeuProLeuAsnAlaGluAlaCysTrpProLysAsp 
151015 
ValGlyIleValAlaLeuGluIleTyrPheProSerGlnTyrValAsp 
202530 
GlnAlaGluLeuGluLysTyrAspGlyValAspAlaGlyLysTyrThr 
354045 
IleGlyLeuGlyGlnAlaLysMetGlyPheCysThrAspArgGluAsp 
505560 
IleAsnSerLeuCysMetThrValValGlnAsnLeuMetGluArgAsn 
65707580 
AsnLeuSerTyrAspCysIleGlyArgLeuGluValGlyThrGluThr 
859095 
IleIleAspLysSerLysSerValLysThrAsnLeuMetGlnLeuPhe 
100105110 
GluGluSerGlyAsnThrAspIleGluGlyIleAspThrThrAsnAla 
115120125 
CysTyrGlyGlyThrAlaAlaValPheAsnAlaValAsnTrpIleGlu 
130135140 
SerSerSerTrpAspGlyArgTyrAlaLeuValValAlaGlyAspIle 
145150155160 
AlaValTyrAlaThrGlyAsnAlaArgProThrGlyGlyValGlyAla 
165170175 
ValAlaLeuLeuIleGlyProAsnAlaProLeuIlePheGluArgGly 
180185190 
LeuArgGlyThrHisMetGlnHisAlaTyrAspPheTyrLysProAsp 
195200205 
MetLeuSerGluTyrProIleValAspGlyLysLeuSerIleGlnCys 
210215220 
TyrLeuSerAlaLeuAspArgCysTyrSerValTyrCysLysLysIle 
225230235240 
HisAlaGlnTrpGlnLysGluAlaAsnAspAsnAspPheThrLeuAsn 
245250255 
AspPheGlyPheMetIlePheHisSerProTyrCysLysLeuValGln 
260265270 
LysSerLeuAlaArgMetLeuLeuAsnAspPheLeuAsnAspGlnAsn 
275280285 
ArgAspLysAsnSerIleTyrSerGlyLeuLysAlaPheGlyAspVal 
290295300 
LysLeuGluAspThrTyrPheAspArgAspValGluLysAlaPheMet 
305310315320 
LysAlaSerSerGluLeuPheSerGlnLysThrLysAlaSerLeuLeu 
325330335 
ValSerAsnGlnAsnGlyAsnMetTyrThrSerSerValTyrGlySer 
340345350 
LeuAlaSerValLeuAlaGlnTyrSerProGlnHisLeuAlaGlyLys 
355360365 
ArgIleGlyValPheSerTyrGlySerGlyLeuAlaAlaThrLeuTyr 
370375380 
SerLeuLysValThrGlnAspAlaThrProGlySerAlaLeuAspLys 
385390395400 
IleThrAlaSerLeuCysAspLeuLysSerArgLeuAspSerArgThr 
405410415 
GlyValAlaGlnAspValPheAlaGluAsnMetLysLeuArgGluAsp 
420425430 
ThrHisHisLeuValAsnTyrIleProGlnGlySerIleAspSerLeu 
435440445 
PheGluGlyThrTrpTyrLeuValArgValAspGluLysHisArgArg 
450455460 
ThrTyrAlaArgArgProThrProAsnAspAspThrLeuAspGluGly 
465470475480 
ValGlyLeuValHisSerAsnIleAlaThrGluHisIleProSerPro 
485490495 
AlaLysLysValProArgLeuProAlaThrAlaAlaGluProGluAla 
500505510 
AlaValIleSerAsnGlyValTrp 
515520 
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