Method to produce high levels of methioninase

The present invention discloses the cloning of a methioninase-encoding nucleic acid molecule from Pseudomonas putida and the construction of high-level expression modules containing the methioninase-encoding nucleic acid molecule. The invention further provides expression modules that use the T7 RNA polymerase promoter to express the isolated methioninase-encoding nucleic acid molecules. Expression modules employing the T7 promoter were found to produce unexpectedly high levels of methioninase. The present invention further provides purification methods to obtain highly pure, endotoxin free methioninase, chemically modified forms of methioninase, crystallized methioninase and lyophilized methioninase preparations. The present invention further provides therapeutical methods using the disclosed recombinant methioninase preparations.

TECHNICAL FIELD 
The present invention relates to expression modules that encode and express 
high levels of recombinant methioninase, recombinant methioninase produced 
using high-level expression modules, compositions containing recombinant 
methioninase produced using high-level expression modules, methods for 
purifying recombinant methioninase produced using high-level expression 
modules, chemically modified forms of recombinant methioninase, and 
methods of using recombinant methioninase produced using high-level 
expression modules in antimethionine and antihomocysteine therapy. 
BACKGROUND 
Therapeutic drug-based treatment of cancer is directed at the use of 
medicinals which selectively inhibit or kill the cancer cells while not 
harming normal tissue function beyond acceptable amounts. The difficulty 
with conventional chemotherapy has been the toxicity of therapeutic drugs 
for normal tissue. 
Many tumors have been shown to have absolute requirement for methionine in 
a variety of cell types and evaluated tumor tissues, including tumors of 
the colon, breast prostate, ovary, kidney, larynx melanoma, sarcoma, lung, 
brain, stomach and bladder as well as leukemias and lymphomas. Methionine 
dependence has been defined as an inability of tumors to grow when 
methionine is replaced by homocysteine in the growth medium. See, for 
example, Chello et al., Cancer Res., 33:1898-1904, 1973; and Hoffman, 
Anticancer Res., 5:1-30, 1985. 
Methionine depletion has been shown to selectively synchronize 
methionine-dependent tumor cells into late S/G.sub.2 phase of the cell 
cycle. Hoffman et al, Proc. Natl. Acad. Sci. USA, 77:7306-7310, 1980. 
Using the combination of methionine deprivation, followed by repletion of 
methionine coupled with exposure to an antimitotic agent, termed 
antimethionine chemotherapy, tumor cells have been selectively eliminated 
from cocultures of normal and tumor cells, resulting in cultures of normal 
cells proliferating vigorously. Stern et al., J. Natl. Cancer Inst., 
76:629-639, 1986. 
However, in order for methionine-dependent chemotherapy to be conducted in 
vivo, it is necessary to have a means to effectively deplete serum of 
circulating methionine. Methionine depletion methods have not been 
described that reduce circulating methionine levels in vivo in a manner 
sufficient to be effective in antitumor therapies. 
Methioninase, an enzyme which degrades methionine, has been purified from a 
variety of bacterial sources, and has been reported to slow the rate of 
tumor cell proliferation in vitro. Kreis et al., Cancer Res., 
33:1862-1865, and 1866-1869, 1973; Tanaka et al., FEBS Letters, 66:307-311 
1976; Ito et al., J. Biochem. 79:1263-1272, 1976; and Nakayama et al., 
Agric. Biol. Chem. 48:2367-2369, 1984. 
Kreis et al., Cancer Res. 33:1866-1869, 1973, have described the use of 
highly impure methioninase preparations isolated from Clostridium 
sporgenes at 1150 units/kg/day to inhibit growth of carcinosarcoma cells 
implanted in a mouse model. Although the enzyme apparently reduced primary 
tumor cell growth, it was not reported to reduce the T/C (treated versus 
control) ratio of tumor diameter below 50%, and was not reported to have 
any effect on metastasis. The authors also indicated that tumor 
specificity of the methioninase cannot be expected without other 
unspecified interventions, and further do not comment on the possibly that 
endotoxin, or other components of the impure preparation, were responsible 
for the effects observed. The only toxicity studies reported were absence 
of animal body weight loss after the duration of the treatment, and 
negative gross examination for toxicity. Further, the authors report that 
the enzyme had a serum half life of 4 hours. 
Kreis et al., Cancer Res. 33:1866-1869, 1973, further reported the use of a 
methionine-free diet as a means to deplete methionine as an antitumor 
therapy. However, the authors reported that the diet did not slow tumor 
growth as effectively as the use of an impure preparation of methioninase 
and resulted in the undesirable side effect of continuous loss of weight 
of the animal. The authors did not report the use of methionine deficient 
diets combined with methioninase treatment, and did not study cell 
synchronization. 
The priority applications of the present invention disclose effective 
chemotherapy of tumors directed at effectively reducing the amount of 
methionine as to provide a beneficial antitumor effect without deleterious 
injury using methioninase. The present invention improves the disclosed 
therapeutic and diagnostic methods and composition by providing a source 
for producing commercially viable quantities of highly pure recombinant 
methioninase. 
BRIEF DESCRIPTION OF THE INVENTION 
The present invention is based, in part, on the generation of high-level 
expression modules encoding methioninase. The expression modules of the 
present invention produce recombinant methioninase in an appropriate host 
cell, such as E. coli, at levels ranging from about 5-75% of total 
cellular protein. 
Based on this observation, the invention provides high expression modules 
encoding methioninase that expresses unexpectedly high levels of 
recombinant methioninase. High expression modules, such as those utilizing 
the T7 RNA polymerase promoter, have been used to produce recombinant 
methioninase at about 1 to 4 gram/liter with a specific activity of about 
2 to 4 units/mg, before purification, using appropriate incubation 
conditions and purification methods. 
The invention further provides methods of accurately selecting 
transformants containing high-level expression modules encoding 
methioninase for the ability to produce high levels of recombinant 
methioninase. Such procedures can be used to specifically select 
transformants containing recombinant methioninase-encoding DNA molecules 
isolated from an organism that naturally produces methioninase, as well as 
to identify transformants that express altered forms of a recombinant 
methioninase-encoding DNA molecule that increases the level of expression 
in a given host or the activity of the recombinant methioninase produced. 
The invention further provides methods of producing recombinant 
methioninase using cells containing high-level expression modules encoding 
methioninase. 
The present invention further provides methods of purifying methioninase to 
obtain a highly pure, endotoxin free methioninase. 
The invention further provides substantially pure recombinant methioninase 
produced using cells containing high-level expression modules encoding 
methioninase. 
The present invention further provides methioninase in crystallized form. 
The invention further provides compositions for diagnostic and therapeutic 
use that contain recombinant methioninase produced using a high-level 
expression module encoding methioninase. 
The invention further provides methods for inhibiting tumor cell growth 
using the recombinant methioninase of the present invention. 
The invention further provides the recombinant methioninase of the present 
invention in chemically modified forms, such as by coupling of the 
recombinant methioninase to polymers such as polyethylene glycol (PEG). 
The recombinant methioninase of the present invention can further be used 
to lower homocysteine levels in patients to reduce the risk of, and to 
treat, cardiovascular diseases. 
The recombinant methioninase of the present invention can further be used 
to deplete methionine for tumor diagnosis and imaging. 
Other features, advantages and related embodiments of the present invention 
will be apparent based on the disclosures contained herein.

DESCRIPTION OF THE INVENTION 
A. Definitions 
"Amino acid residue" refers to an amino acid formed upon chemical digestion 
(hydrolysis) of a polypeptide at its peptide linkages. The amino acid 
residues described herein are preferably in the "L" isomeric form. 
However, residues in the "D" isomeric form can be substituted for any 
L-amino acid residue, as long as the desired functional property is 
retained by the polypeptide. NH2 refers to the free amino group present at 
the amino terminus of a polypeptide. COOH refers to the free carboxy group 
present at the carboxy terminus of a polypeptide. In keeping with standard 
polypeptide nomenclature (described in J. Biol. Chem. 243:3552-59, 1969, 
and adopted at 37 CFR 1.822(b)(2)), hereby incorporated by reference. 
______________________________________ 
TABLE OF CORRESPONDENCE 
SYMBOL AMINO ACID 
1-Letter 3-Letter 
______________________________________ 
Y Tyr tyrosine 
G Gly glycine 
F Phe phenylalanine 
M Met methionine 
A Ala alanine 
S Ser serine 
I Ile isoleucine 
L Leu leucine 
T Thr threonine 
V Val valine 
P Pro proline 
K Lys lysine 
H His histidine 
Q Gln glutamine 
E Glu glutamic acid 
Z Glx Glu and/or Gln 
W Trp tryptophan 
R Arg arginine 
D Asp aspartic acid 
N Asn asparagine 
B Asx Asn and/or Asp 
C Cys cysteine 
J Xaa Unknown or other 
______________________________________ 
It should be noted that all amino acid residue sequences represented herein 
by formulae have a left-to-right orientation in the conventional direction 
of amino terminus to carboxy terminus. In addition, the phrase "amino acid 
residue" is broadly defined to include the amino acids listed in the Table 
of Correspondence and modified and unusual amino acids, such as those 
listed in 37 CFR 1.822(b)(4), and incorporated herein by reference. 
Furthermore, it should be noted that a dash at the beginning or end of an 
amino acid residue sequence indicates a peptide bond to a further sequence 
of one or more amino acid residues or a covalent bond to an amino-terminal 
group such as NH.sub.2 or acetyl or to a carboxy-terminal group such as 
COOH. 
"Recombinant DNA (rDNA) molecule" refers to a DNA molecule produced by 
operatively linking two DNA segments. Thus, a recombinant DNA molecule is 
a hybrid DNA molecule comprising at least two nucleotide sequences not 
normally found together in nature. rDNA's not having a common biological 
origin, i.e., evolutionarily different, are said to be "heterologous". 
"Vector" refers to a rDNA molecule capable of autonomous replication in a 
cell and to which a DNA segment, e.g., gene or polynucleotide, can be 
operatively linked so as to bring about replication of the attached 
segment. Vectors capable of directing the expression of genes encoding for 
one or more polypeptides are referred to herein as "expression vectors". 
Particularly important vectors allow convenient expression of a 
recombinant methioninase protein of this invention. 
B. DNA Segments and Vectors 
1. Methioninase-Coding DNA Molecules 
It has been found that by operably linking an isolated DNA molecule 
encoding methioninase to a promoter, particularly an RNA polymerase 
promoter such as the T7 RNA polymerase promoter, recombinant methioninase 
can be expressed at levels from about 5-75% of total cellular protein, 
when introduced into an appropriate host cell. Accordingly, the invention 
provides high-level expression modules that express high levels of 
recombinant methioninase when introduced into a host under appropriate 
conditions. 
As used herein, a high-level expression module, or an expression module of 
the present invention, refers to a nucleic acid molecule that contains one 
or more expression control elements that direct the transcription and 
translation of an operably linked nucleotide sequence that encodes 
methioninase. The expression module can be an isolated nucleic acid 
molecule or can be present in a vector (described below). 
The expression modules of the present invention contain control elements 
that direct the production of recombinant methioninase such that the 
recombinant methioninase produced represents from about 5-75% of total 
cellular protein, preferably more than 10% of total cellular protein. The 
preferred expression control elements are RNA polymerase promoters, the 
most preferred being the T7 RNA polymerase promoter. Other examples of RNA 
polymerase promoters include, but are not limited to, the Tac and Trc 
promoters. 
A promoter is an expression control element formed by a DNA sequence that 
permits binding of RNA polymerase and transcription to occur. Promoter 
sequences compatible with a particular hosts system are known in the art 
and are typically provided in a plasmid vector containing one or more 
convenient restriction sites. Typical of such plasmids vectors are those 
containing the T7 RNA polymerase promoter, pT7 and pET that are available 
from a variety of sources such as commercial suppliers and the American 
Type Culture Collection. 
The expression modules of the present invention further comprise a nucleic 
acid sequence that encodes methioninase. As used herein, a nucleic acid 
sequence is said to encode methioninase when the transcription and 
translation of the nucleic acid molecule comprising the sequence results 
in the production of a protein having methioninase activity. 
L-Methioninase (L-methionine-alpha-deamino-gammamercaptomethane-lyase or 
methioninase) is an enzyme that degrades methionine by deamination and 
dethiomethylation. Methioninase activity can be measured at least by 
measuring the amount of alpha-ketobutyrate formed upon cleavage of 
methionine. One unit (U) of methioninase is defined as an amount of enzyme 
that produces 1 micromole of alpha-ketobutyrate per minute from methionine 
under the standard assay conditions described by Ito et al., J. Biochem., 
79:1263-1272, 1976; and Soda, Analyt. Biochem. 25:228-235, 1968. 
The methioninase-encoding nucleic acid sequence can comprise an unaltered 
sequence obtained from an organism that naturally produces recombinant 
methioninase, or can comprise a sequence obtained from an organism that 
naturally produces methioninase that has been altered to contain one or 
more nucleic acid or amino acid substitutions, deletions or additions. 
The methioninase-encoding nucleic acid molecule, whether altered or 
unaltered, can be derived from any organism that naturally produces 
methioninase. The preferred source of the methioninase-encoding nucleic 
acid molecule is Pseudomonas putida. Example 1 discloses the isolation and 
sequencing of a methioninase-encoding nucleic acid molecule from P. 
putida. Other preferred sources for a methioninase-encoding nucleic acid 
molecule include, but are not limited to, Trichomonas vaginalis, 
Nippostrongylus brasiliensis, and Fusobacterium sp. 
The complete coding sequence for methioninase can be obtained from a 
variety of sources, especially those recited above, using a variety of 
methods. The isolation of methioninase-encoding nucleic acid molecules 
from an organism other than P. putida is greatly facilitated by the amino 
acid and nucleic acid sequences provided in SEQ ID NO:1. 
Specifically, a skilled artisan can readily use the nucleic acid sequence 
provided in Seq. ID NO:1 to prepare pairs of oligonucleotide primers for 
use in a polymerase chain reaction (PCR) to selectively amplify a 
methioninase-encoding nucleic acid molecule from methionine expressing 
organisms. The preferred PCR primer pairs based on the sequence provided 
in SEQ ID NO:1 are: 
5'-GCCGGTCTGTGGAATAAGCT-3' (Sense) SEQ ID NO:4 
5'-CCAGGGTCGACTCCAGCGCC-3' (Antisense) SEQ ID NO:5 
A preferred PCR denature/anneal/extend cycle for using the above PCR 
primers is as follows: first denaturation at 95.degree. C. for 10 minutes, 
then 5 cycles of denaturation at 94.degree. C. for 30 seconds, annealing 
at 60.degree. C. for 30 seconds, and extension at 72.degree. C. for 2 
minutes; then 25 cycles of denaturation at 94.degree. C. for 30 seconds, 
60.degree. C. for 30 seconds, then extension at 72.degree. C. for 1.5 
minutes; then final extension at 72.degree. C. for 10 minutes. The PCR 
amplified products are two bands of which the 1365 bp band was collected, 
and purified as the insert ONCase-1 DNA. 
Alternatively, a fragment of the nucleotide sequence or SEQ. ID No. 1 can 
be used as a probe to isolate DNA encoding methioninase from organisms 
other than Pseudomonas putida using art-known methods. Oligomers 
containing approximately 18-20 nucleotides (encoding about a 6-7 amino 
acid stretch) are prepared and used to probe genomic DNA libraries to 
obtain hybridization under conditions of sufficient stringency to 
eliminate false positives using procedures well known in the art. (See 
Sambrook et al. Molecular Cloning, Cold Spring Harbor Press 1989) 
DNA segments (i.e., synthetic oligonucleotides) that are used as probes or 
specific primers for the polymerase chain reaction (PCR), as well as gene 
sequences encoding methioninase, can readily be synthesized by chemical 
techniques, for example, the phosphotriester method of Matteucci, et al., 
(J. Am. Chem. Soc. 103:3185-3191, 1981) or using automated synthesis 
methods. In addition, larger DNA segments can readily be prepared by well 
known methods, such as synthesis of a group of oligonucleotides that 
define the DNA segment, followed by hybridization and ligation of 
oligonucleotides to build the complete segment. 
In addition to PCR and DNA probe based methods, DNA molecules encoding 
methioninase can be isolated using polyclonal antiserum or monoclonal 
antibodies raised against peptide fragments of SEQ ID NO:1 that are 
predicted as being immunogenic. Such antibodies can be used to probe an 
expression library generated from a given organism, such as a lambda gtll 
library, to obtain DNA molecules encoding methioninase from an organism 
other the P. putida. 
Once a naturally occurring methioninase-encoding nucleic acid molecule is 
obtain, a skilled artisan can readily employ random or site specific 
mutagenesis procedures to alter the methioninase-encoding sequence so as 
to increase the level of expression or to substitute, add, or delete one 
or more amino acids from the encoded methioninase. 
In one embodiment, the methioninase-encoding sequence is altered so as to 
increase the level of expression of the recombinant methioninase in a 
given host cell without changing the amino acid sequence of the encoded 
methioninase. Increased expression of recombinant methioninase in a 
particular host can be obtained by altering one or more of the codons 
present in the nucleic acid molecule so that the resulting codons are ones 
that are more frequently used by the host organism to encode a particular 
amino acid. Altering a nucleotide sequence to contain preferred codons can 
be accomplished using art known procedures such as site directed 
mutagenesis or by synthesizing a nucleic acid molecule containing the 
preferred codons. 
In addition to alterations that affect expression, methioninase-encoding 
nucleic acid molecules can be altered so as to facilitate purification of 
the resulting protein. For example, as disclosed in the Examples, by 
altering either the amino or carboxy terminus of the recombinant 
methioninase so as to add a polyhistidine stretch, Ni.sup.++ sepharose 
can be used to purify the resulting fusion protein. 
The methioninase-encoding sequence can also be altered to introduce changes 
in the amino acid sequence of the encoded methioninase, so as to add, 
substitute, or delete one or more amino acid residues. The resulting 
recombinant methioninase will preferably contain alterations that result 
in recombinant methioninase with better biological or physiological 
properties such as increased activity, decreased immunogenicity, or 
increased serum half life. Such altered forms can be rationally designed 
or randomly generated. 
An alteration is said to be rationally designed when the alteration is 
specifically chosen based on the amino acid sequence of the starting and 
resulting proteins and a desired physiological property. For example, one 
type of rationally designed alteration is to replace hydrophobic amino 
acids with less hydrophobic residues to increase solubility. The preferred 
method for generating rationally designed alterations is site direct 
mutagenesis using a mismatched PCR primer extension method. 
Alterations are said to be randomly generated when the alteration is not 
rationally selected. Random mutagenesis techniques, such as chemical 
mutagenesis and linker scanning mutagenesis, generate a large variety of 
random and non-specific alterations in a given protein encoding sequence. 
Such methods can be used to radically alter the methioninase-encoding 
nucleic acid molecule. 
Altered forms of recombinant methioninase generated in this fashion are 
then screened for desired properties using a variety of art known methods. 
The choice of selection method employed will be dependent on the host, 
vector, and mutagenesis methods employed as well as the properties that 
are selected for. 
The present invention further provides vectors containing one or more of 
the expression modules of the present invention. Vectors are DNA molecules 
that are capable of autonomous replication within a host. Vectors can 
contain an episomal origin of replication derived from a naturally 
occurring plasmid, a genomic origin of replication, or can be derived from 
a viral genome. The choice of the vector to which an expression module of 
the present invention is inserted depends directly, as is well known in 
the art, on the functional properties desired, e.g., protein expression, 
and the host cell to be transformed. 
In one embodiment, the vector includes a prokaryotic replicon. Prokaryotic 
replicons such as the ColEl replicon, are well known in the art and can 
readily be employed in combination with an expression module of the 
present invention. In addition, the vector may include a gene encoding a 
selectable marker such as a drug resistance. 
Eukaryotic expression vectors can also be used in combination with an 
expression module of the present invention. Eukaryotic cell expression 
vectors are well known in the art and are available from several 
commercial sources. Typical of such vectors are PSVL and pKSV-10 
(Pharmacia), pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 
(ATCC, #31255), the vector pCDM8 described herein, and the like eucaryotic 
expression vectors. High level expression vectors can further be generated 
using insect cell expression systems such as a bacculovirus based vector 
system. 
In general terms, the generation of a high expression module encoding 
methioninase typically involves the following: 
First, a DNA is obtained that encodes methioninase. If the sequence is 
uninterrupted by introns, as expected from a bacterial source, it is 
suitable for expression in any host. This sequence may be altered to be in 
a readily excisable and recoverable form by inserting sequences containing 
one or more restriction endonuclease sites at regions flanking the 
methioninase-encoding sequence. 
The excised or recovered coding sequence is then placed in operable linkage 
with a high expression control element, preferably in a replicable 
expression vector. The expression module or vector is then used to 
transform a suitable host and the transformed host is cultured under 
conditions to effect the production of the recombinant methioninase. 
Optionally the recombinant methioninase is isolated from the medium or 
from the cells; recovery and purification of the protein may not be 
necessary in some instances, where some impurities may be tolerated. 
Each of the foregoing steps can be done in a variety of ways. For example, 
the desired coding sequences may be obtained from genomic fragments and 
used directly in appropriate hosts. The constructions of expression 
vectors that are operable in a variety of hosts are made using two or more 
appropriate replicons and control elements. Suitable restriction sites 
can, if not normally available, be added to the ends of the coding 
sequence so as to provide an excisable gene to insert into these vectors. 
3. Transformed Host Cells Expressing High Levels of Recombinant 
methioninase 
The present invention further provides host cells transformed with an 
expression module or vector of the present invention so as to produce from 
about 5-75% of total cellular protein as recombinant methioninase, 
preferably more than about 10% of total cellular protein. The host cell 
can be either a prokaryotic or a eucaryotic host. 
Any prokaryotic host can be used to express the high-level 
methioninase-encoding modules of the present invention. The preferred 
prokaryotic host is E. coli. In the Examples that follow, the DH5.alpha. 
and BL21(DE3) strains of E. coli were used. 
Preferred eucaryotic host cells include insect cells, yeast cells and 
mammalian cells, preferably insect cells such as SP6 and vertebrate cells 
such as those from a mouse, rat, monkey or human fibroblastic cell line. 
Other preferred eucaryotic host cells include Chinese hamster ovary (CHO) 
cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells 
NIH/3T3 available from the ATCC as CRL 1658, baby hamster kidney cells 
(BHK), and the like eucaryotic tissue culture cell lines. 
Transformation of an appropriate host with a high-level expression 
recombinant module of the present invention is accomplished by well known 
methods that typically depend on the type of host and vector used. With 
regard to transformation of prokaryotic host cells, electroporation or 
salt treatment of the host cells is preferred, for example, see Cohen et 
al., Proc. Natl. Acad. Sci. USA 69:2110, 1972; and Maniatis et al., 
Molecular Cloning, A Laboratory Mammal, Cold Spring Harbor Laboratory, 
Cold Spring Harbor, N.Y. (1982). 
With regard to transformation of eukaryotic cells, electroporation or the 
use of a cationic lipid is preferred, for example, see Graham et al., 
Virol. 52:456, 1973; and Wigler et al., Proc. Natl. Acad. Sci. USA 
76:1373-76, 1979. 
Successfully transformed cells, i.e., cells that contain an expression 
module of the present invention, can be identified by well known 
techniques. For example, cells resulting from the introduction of an 
expression module of the present invention can be cloned to produce single 
colonies. Cells from those colonies can be harvested, lysed and their DNA 
content examined for the presence of the rDNA using a method such as that 
described by Southern, J. Mol. Biol., 98:503, 1975, or Berent et al., 
Biotech. 3:208, 1985. However, as described below, the present invention 
further provides a rapid screening method to identify transformants which 
express high levels of recombinant methioninase. 
4. Identification of Hosts Expressing High Levels of Recombinant 
methioninase 
The present invention further provides methods of identifying a transformed 
host cell which produces recombinant methioninase at levels from about 
5-75% of total cellular protein. Specifically, it has been observed that 
transformed host cells expressing from about 5-75% of total cellular 
protein as recombinant methioninase, have a distinct and observable pink 
color. This is particularly pronounced when E. coli is used as the host. 
To identify a transformed host cell expressing high levels of recombinant 
methioninase, a transformed cell is grown on or in a media under 
conditions in which the recombinant methioninase is expressed and that 
allows visual inspection of the growing cells. The growing cells or 
colonies are examined and selected based on the displaying of a pink 
color. 
A variety of culture/growth conditions can be employed to grow transformed 
host cells for selection using the present methods. The components of the 
growth medium will depend on the nutritional requirements of the 
host/vector system employed and inspection system used to identify the 
pink color associated with high levels of recombinant methioninase 
expression and thereby allowing isolation of a high-level expression 
clone. The preferred medium is a solid medium onto which the transformed 
host cells can be plated and grown as isolated colonies, each of which is 
derived from single host. The preferred method of identifying high-level 
expression clones is visual inspection of growing colonies. 
5. Production of Recombinant methioninase Using a High-level expression 
module 
The present invention further provides methods for producing recombinant 
methioninase. Specifically, recombinant methioninase can be produced at 
commercially significant levels using a host transformed with one or more 
of the high-level expression modules of the present invention. Such a 
transformed host will express recombinant methioninase at a level from 
about 5-75% of total cellular protein. Using the hosts of the present 
invention, a skilled artisan can readily produce recombinant methioninase 
for use in a variety of diagnostic and therapeutic methods using art known 
methods. 
The preferred method for purifying recombinant methioninase produced using 
a transformed host containing a high expression module encoding 
methioninase comprises the steps of: 
a) heating an extract of a transformed cell that contains methioninase in 
aqueous buffers from about 40.degree.-60.degree. C. for about 1-10 min., 
preferably 50.degree. C. for 1 min.; 
b) centrifugation of the heated extract from about 10k to 20k rpm in a GS-3 
rotor (Sorvall, Du Pont) for about 15 min. to 1 hour, preferably at about 
13K rpm for about 30 min. at 4.degree. C.; 
c) ultrafiltration of the supernatant using a filter of about 50K to 100K 
pore size, preferably a Millipore Pre/Scale:TFF PLHK 100K 2.5 ft.sup.2 
cartridge using a 10 mM potassium phosphate buffer (pH8.3); 
d) DEAE ion exchange chromatography in low ionic strength (from about 10-50 
mM) KCl in a 10-20 mM potassium phosphate buffer at about pH 7.0-7.6, and 
collecting fractions containing methioninase eluted in a 40-200 mM KCl 
gradient, preferably using DEAE-Sepharose FF column; 
e) a second DEAE ion exchange chromatography in medium ionic strength 
(50-100 mM) KCl in a 10-20 mM potassium phosphate buffer at about pH 
8.0-8.6, and collecting fractions containing methioninase eluted in a 
phosphate buffer (pH 8.3) eluted in 100-200 mM KCl, preferably using 
DEAE-Sepharose FF column; and 
f) contacting said fractions collected in step (e) with a chromatography 
medium capable of absorbing endotoxin, and collecting the eluant, thereby 
removing endotoxin from said eluant to form endotoxin-free methioninase 
having at least 20 units methioninase activity per milligram protein and 
from 1-100 ng of endotoxin per mg protein, preferably using an 
Acticlean.RTM. Etox column. 
The cell extract is prepared from a host cell that has been altered to 
express high levels of recombinant methioninase (from about 5-75% of total 
cellular protein). For bacterial cell extracts, the extracts are generally 
prepared by first harvesting and washing bacterial cell cultures to form a 
cell paste/pellet, depending upon whether harvesting is by centrifugation 
or by hollow fiber filtration, which methods are generally well known. 
The cells are then disrupted using conventional means. Preferably the cells 
are disrupted using a homogenizer, such as a cavitator-type homogenizer, 
for example, a Microfluidics Corp. Model #HC8000. 
The resulting suspension is heated to precipitate selective proteins and 
other insoluble materials. Typical heating conditions are from about 
45.degree.-60.degree. C. for 1-10 minutes. Preferred is a heating step of 
50.degree. C. for 1 minute. 
The heated extract is centrifuged to remove debris, and the supernatant is 
filtered and applied to DEAE ion-exchange chromatography medium in two 
steps as described above. Preferred adsorption and elution conditions are 
described in the Examples. Any of a variety of DEAE ion exchange column 
chromatography media can be used in these steps, and the choice of media 
is not to be construed as limiting. Commercial sources include Pharmacia 
Fine Chemicals, BioRad, and Sigma. 
Thereafter, endotoxin is removed to produce a protein having acceptable 
levels of endotoxin as recited earlier. The endotoxin removal step can be 
carried out in any of a variety of means, as are well known, and typically 
involve contacting the protein in solution with a chromatography medium 
capable of adsorbing endotoxin, and yielding a chromatography medium 
eluant which contains endotoxin-free protein. The preferred commercial 
reagent for use in removing endotoxin is Acticlean.RTM. Etox. 
C. Therapeutic Compositions 
The present invention further provides therapeutic compositions comprising 
a therapeutically effective amount of substantially isolated recombinant 
methioninase that is produced using a host transformed with a high-level 
expression module encoding methioninase. 
The compositions of the present invention will preferable contain 
recombinant methioninase that has a specific activity of about 10 to 50 
units (U) per mg protein. Typical preparations of purified recombinant 
methioninase are described herein having a specific activity of about 16 
tp 24 U/mg. In the Examples, recombinant methioninase prepared using the 
expression vector pAC-1 had a specific activity of 20.1 U/mg. 
The recombinant methioninase in the compositions of the present invention 
is preferably substantially isolated. By substantially isolated is meant 
that the enzyme is at least 90% pure by weight, preferably at least 95% 
pure, and more preferably at least 99% pure, or essentially homogeneous. A 
preferred recombinant methioninase is essentially homogeneous when 
analyzed on electrophoretic media such as polyacrylamide gel 
electrophoresis (PAGE or SDS-PAGE). Homogeneous on PAGE means only a 
single detectable band. 
The recombinant methioninase used to prepare the compositions of the 
present invention is preferably substantially free of endotoxins, such as 
bacterial lipopolysaccharides, due to the undesirable side effects 
associated with endotoxins when physiologically contacted in a mammal, as 
by i.v. or i.p. administration. By substantially free is meant less than 
about 10 nanograms (ng) endotoxin per milligram (mg) recombinant 
methioninase protein, preferably less than 1 ng endotoxin per mg 
recombinant methioninase, and more preferably less than 0.1 ng endotoxin 
per mg recombinant methioninase. 
The recombinant methioninase used to prepare the compositions of the 
present invention is preferably prepared from a gene cloned from P. putida 
and expressed using a high-level expression vector as herein described. 
The recombinant methioninase containing compositions of the present 
invention may further comprise a physiologically tolerable carrier. As 
used herein, the terms "pharmaceutically acceptable", "physiologically 
tolerable" and grammatical variations, both referring to compositions, 
carriers, diluents and reagents that the materials are capable of 
administration to or upon a mammal or human without the production of 
undesirable physiological effects such as nausea, dizziness, gastric upset 
and the like. 
The preparation of a pharmacological composition that contains active 
ingredients dissolved or dispersed therein is well understood in the art. 
Typically such compositions are prepared as sterile injectables either as 
liquid solutions or suspensions, aqueous or non-aqueous, however, solid 
forms suitable for solution, or suspensions, in liquid prior to use can 
also be prepared. The preparation can also be emulsified. In addition, a 
therapeutic amount of recombinant methioninase can be present in a 
ointment or on a diffusible patch, such as a bandage, as to afford local 
delivery of the agent. 
The active ingredient can be mixed with excipients which are 
pharmaceutically acceptable and compatible with the active ingredient and 
in amounts suitable for use in the therapeutic methods described herein. 
Suitable excipients are, for example, water, saline, dextrose, glycerol, 
or the like and combinations thereof. In addition, if desired, the 
composition can contain minor amounts of auxiliary substances such as 
wetting or emulsifying agents, pH buffering agents and the like which 
enhance the effectiveness of the active ingredient. 
The therapeutic composition of the present invention can include 
pharmaceutically acceptable salts of the components therein. 
Pharmaceutically acceptable salts include the acid addition salts (formed 
with the free amino groups of the polypeptide) that are formed with 
inorganic acids such as, for example, hydrochloric or phosphoric acids, or 
such organic acids as acetic, tartaric, mandelic and the like. Salts 
formed with the free carboxyl groups can also be derived from inorganic 
bases such as, for example, sodium, potassium, ammonium, calcium or ferric 
hydroxides, and such organic bases as isopropylamine, trimethylamine, 
2-ethylamino ethanol, histidine, procaine and the like. 
Physiologically tolerable carriers are well known in the art. Exemplary of 
liquid carriers are sterile aqueous solutions that contain no materials in 
addition to the active ingredients and water, or contain a buffer such as 
sodium phosphate at physiological pH value, physiological saline or both, 
such as phosphate-buffered saline. Still further, aqueous carriers can 
contain more than one buffer salt, as well as salts such as sodium and 
potassium chlorides, dextrose, propylene glycol, polyethylene glycol and 
other solutes. 
Liquid compositions can also contain liquid phases in addition to and to 
the exclusion of water, as described herein. Exemplary of such additional 
liquid phases are glycerin, vegetable oils such as cottonseed oil, organic 
esters such as ethyl oleate, and water-oil emulsions, particularly the 
liposome compositions described earlier. 
A therapeutic composition contains an effective amount of recombinant 
methioninase, typically an amount of at least 0.1 weight percent of active 
protein per weight of total therapeutic composition, and preferably is at 
least about 25 weight percent. A weight percent is a ratio by weight of 
recombinant methioninase protein to total composition. Thus, for example, 
0.1 weight percent is 0.1 grams of recombinant methioninase per 100 grams 
of total composition. 
Insofar as a recombinant methioninase composition can be used in vivo 
intravascularly, it is contemplated in one embodiment to formulate a 
therapeutic composition for controlled delivery of the recombinant 
methioninase, and optionally to shield the recombinant methioninase 
protein from degradation and other phenomenon which would reduce the serum 
half-life of therapeutically administered recombinant methioninase. 
Thus, in one embodiment, the invention contemplates therapeutic 
compositions containing delivery vehicles such as polymers, polymeric 
vehicles, particulates, latexes, coacervates, ion-exchange resins, 
liposomes, enteric coatings, mediators, bioadhesives, microcapsules, 
hydrogels, and the like vehicles. Exemplary drug delivery vehicles 
including liposomes are described at least by Tarcha in "Polymers For 
Controlled Drug Delivery", CRC Press, Boca Raton, 1990. 
D. Chemically Modified Recombinant methioninase 
The present invention further provides the recombinant methioninase of the 
present invention that is chemically modified, for example by conjugation 
to a polymer. By "chemically modified" is meant any form of recombinant 
methioninase that is changed to a form that is different than the 
recombinant methioninase purified from nature. Preferably, the recombinant 
methioninase is chemically modified by linking the recombinant 
methioninase to a polymer or to a polyalkylene oxide. Recombinant 
methioninase conjugated to a polymer increases the serum half-life and 
decreases the immunogenicity or antigenicity of the resulting compound. 
Examples of polymers and polyalkylene oxide to which proteins may be 
attached include, but are not limited to, polyethylene glycol, 
particularly MSC-5000 PEG, polyethylene oxide, polypropylene oxide, 
copolymers of ethylene oxide, and copolymers of propylene oxide. Methods 
for chemically modifying proteins are well known to the art and can 
readily be used to modify the recombinant methioninase of the present 
invention, for example, see priority application PCT/US93/11311. 
E. Formulations of recombinant methioninase 
The present invention further provides methioninase in lyophilized or 
crystalline form. In detail, it has been observed that methioninase can 
readily be lyophilized or crystallized using art known methods. The 
resulting preparation of methioninase, crystallized or lyophilized forms, 
were found to be highly stable, readily hydratable, and remained highly 
active following rehydration. 
A variety of art known methods can be used to obtain methioninase in 
crystallized or lyophilized form. In the examples, lyophilization and 
crystallization of methioninase were performed using a Verdis, Freeze 
mobile 24, at 100 milifar, -80.degree. C. for 72 hours. A skilled artisan 
can readily adapt other art known procedures for use in producing 
lyophilized or crystallized forms of methioninase. 
F. Uses for the Recombinant methioninase of the Present Invention 
The recombinant methioninase of the present invention can be used in 
diagnostic and therapeutic methods that have been developed and described 
elsewhere that use methioninase purified from a natural sources, see 
PCT/US93/11311. For example, the recombinant methioninase of the present 
invention can be used 1) as an antitumor agent in a variety of modalities, 
such as by depleting methionine from a tumor cell, which are possibly 
universally methionine dependent, tumor tissue or the circulation of a 
mammal with cancer, so that the tumor growth will be inhibited 2) to 
induce cell cycle stasis in tumor cells followed by cell synchronization 
and the use of antimitotic agents, 3) in combination with antimitotic and 
cell cycle-specific cytotoxic agents, 4) to deplete cellular methionine 
prior to labeling with .sup.11 C} methionine, which can be used in tumor 
diagnosis and localization, 5) to deplete serum homocysteine to prevent 
and cure cardiovascular diseases that are mediated by high serum levels of 
homocysteine. In the Examples that follow, the recombinant methioninase of 
the present invention was administered to three patients. Infusion dosage 
of up to 20,000 units, infused over ten hours, had no significant side 
effects and yielded a depletion of methionine for 10 hours following 
infusion. A skilled artisan will readily use the recombinant methioninase 
of the present invention as a substitute for recombinant methioninase 
derived from other sources in any art-known method of use. 
The following examples relating to this invention are illustrative and 
should not, of course, be construed as specifically limiting the 
invention. Moreover, such variations of the invention, now known or later 
developed, which would be within the purview of one skilled in the art are 
to be considered to fall within the scope of the present invention 
hereinafter claimed. 
EXAMPLE 1 
Isolation of Nucleic Acid Molecules Encoding Methioninase PCR Reaction of 
the Insert of Methioninase Gene Clone: 
Genomic DNA of Pseudomonas putida AC-1, derived from ATCC8209, was used as 
template; the primers used were as follows: 
t1:5'-GCCGGTCTGTGGAATAAGCT-3' (Sense), (SEQ ID NO:4) HindIII 
t2:5'-CCAGGGTCGACTCCAGCGCC-3' (Antisense). (SEQ ID NO:5) Sal I 
The PCR reaction condition was as follows: first denaturation at 95.degree. 
C. for 10 minutes, then 5 cycles of denaturation at 94.degree. C. for 30 
seconds, annealing at 60.degree. C. for 30 seconds, and extension at 
72.degree. C. for 2 minutes; then 25 cycles of denaturation at 94.degree. 
C. for 30 seconds, 60.degree. C. for 30 seconds, then extension at 
72.degree. C. for 1.5 minutes; then final extension at 72.degree. C. for 
10 minutes. The PCR amplified products are two bands of which the 1365 bp 
band was collected, and purified as the insert ONCase-1 DNA. 
Cloning and Transformation 
The ONCase-1 DNA was ligated with pT7Blue T-vector (Novagen) at the EcoR V 
T-cloning site. The pONCase-1 DNA was transformed into DH5-.alpha. 
bacterial cells using standard procedures. 
DNA Sequencing 
DNA sequencing was performed using T7 DNA polymerase and the dideoxy 
nucleotide termination reaction. The primer walking method was used. 
.sup.35 S! DATP was used for labeling. Sequencing reactions were analyzed 
on 6% polyacrylamide wedge or non-wedge gels containing 8M urea. DNA 
samples were loaded in the order of ACGT. DNA sequences were analyzed by 
MacVector. The DNA sequence and corresponding amino acid sequence are 
provided in FIG. 1. 
EXAMPLE 2 
High Expression Clones of Recombinant Methioninase PCR Reaction of the 
Insert for the Methioninase Expression Clone: 
The pONCase-1 clone was used as the template, the primers used are as 
follows: 
t14. 5'-GGAATTCCATATGCACGGCTCCAACAAGC-3' (Sense) (SEQ ID NO:6) NdeI 
t15. 5'-AGTCATGGATCCTCATCATCATCATCATCATGGCACTCGCCTTGAGTGC-3' BamHI 
(Antisense) SEQ ID NO:7) 
t18. 5'-AGTCATCCTAGGTCAGGCACTCGCCTTGAGTGC-3' (Antisense) BamHI (SEQ ID 
NO:7) 
The PCR reaction condition was as follows: first denaturation at 95.degree. 
C. for 10 minutes, then 5 cycles of denaturation at 94.degree. C. for 1 
minute, annealing at 56.degree. C. for 1.5 minutes, and extension at 
72.degree. C. for 2 minutes; then 20 cycles of denaturation at 94.degree. 
C. for 30 seconds, 56.degree. C. for 30 seconds, then extension at 
72.degree. C. for 1.5 minutes; then final extension at 72.degree. C. for 
10 minutes. Two PCR amplified products, ONCase-2 (1238 bp), ONCase-3 (1220 
bp) band were collected and purified. 
Cloning and Transformation 
The DNA of ONCase-2 and ONCase-3 DNA was digested with NdeI and BamHI and 
ligated with the pT7.7 vector at the NdeI and BamHI cloning sites. The 
pONCase-2 and pONCase-3 DNA sequences were then transformed into BL21 
(DE3) bacterial cells using standard procedures. 
Selection of pAC-1 and pAC-2 Clones 
The positive clones were selected from Ampicillin-containing plates. After 
storage at 4.degree. C. for 24 hours, the positive clones which expressed 
high level of recombinant methioninase had a distinct pink color that 
allowed their identification and selection. The methioninase expression 
levels of the positive clones were determined by activity assay. Two high 
expression clones were selected as the pAC-1 clone which contained 
ONCase-3 and as the pAC-2 clone which contained ONCase-2. 
Construction of pAC-3 Clone and pAC-4 Clone 
The tetracycline resistance gene was obtained from pBR322 at the Ava I and 
Cla I sites. The Ava I end was filled into a blunt end, and was ligated 
with pAC-1 which was digested with the BamH I and Cla I restriction 
enzymes, with the BamH I end filled into a blunt end. Positive clones 
which became pink after storage at 4.degree. C. for 24 hours were selected 
from Tetracycline-containing plates. A high expression recombinant 
methioninase clone was determined by activity assay and named as the pAC-3 
clone. 
The Tetracycline-resistance gene was also obtained from pBR322 at the Ava I 
and Hind III sites. The Ava I end was filled into a blunt end, and was 
ligated with pAC-1 which was digested with the Hind III and Cla I 
restriction enzymes, with the Cla I end filled into a blunt end. Positive 
clones which became pink after storage at 4.degree. C. for 24 hours were 
selected from Tetracycline-containing plates. A high expression 
recombinant methioninase clone was determined by activity assay and named 
as the pAC-4 clone. A variety of high level expression clones are provided 
in Table 1. 
TABLE 1 
______________________________________ 
rMETase Expression Clones 
Antibiotic Expression* 
Clone Vector Resistance 
Promoter 
Fusion (g/1) 
______________________________________ 
pAC-1 pT7.7 Amp T7 -- 1.0 
pAC-2 pT7.7 Amp T7 His. Tag 
0.5 
pAC-3 pT7.7 Tc T7 -- 0.5 
pAC-4 pT7.7 Tc T7 -- 1.0 
______________________________________ 
*Expression level in shaking flask (TB medium, 37.degree. C., 400 rpm, 36 
hours). 
EXAMPLE 3 
Fermentation of Recombinant Methioninase Expression Clones 
The expression clones of recombinant methioninase were grown in Terrific 
Broth medium containing either Ampicillin (100 .mu.g/ml) or Tetracycline 
(10 .mu.g/ml), at 28.degree. C. or 37.degree. C. with 400 rpm shaking in a 
6-L flask or fermenter. 
EXAMPLE 4 
Purification of Recombinant Methioninase 
An outline of the purification method is provided in FIGS. 2 and 3. 
(1) Pre-column treatment of the sample 
The bacteria were harvested by centrifugation at 800.times.g at 4.degree. 
C. for 10 min. The bacterial pellet is then suspended in extraction 
solution (20 mM potassium phosphate pH9.0, 10 .mu.M pyridoxal phosphate 
and 0.01% .beta.-mercaptoethanol) and disrupted with a cavitator-type 
homogenizer (Microfluidics Corp. model # HC 8000). Heat treatment of the 
homogenate is then carried out at 50.degree. C. for one minute. The 
suspension is centrifuged with an automatic refrigerated centrifuge 
(SORVALL Superspeed RC 2-B) at 4.degree. C. at 13k rpm for 30 min. The 
supernatant is then collected. This step is followed by ultrafiltration by 
a Millipore Prep/Scale--TFF PLHK 100k 2.5 ft.sup.2 cartridge with buffer 
(10 mM potassium phosphate pH8.3). The pH is adjusted to 7.2 by 
ultrafiltration. 
(2) Chromatographic conditions 
The first column: DEAE Sepharose FF 
Column: XK 100/60, Height: 32 cm, Volume: 2.5 L 
Solution: A! 40 mM potassium chloride, 10 mM potassium phosphate (pH7.2) 
containing 10 .mu.M pyridoxal phosphate and 0.01% .beta.-mercaptoethanol. 
B! 200 mM potassium chloride, 10 mM potassium phosphate (pH7.2) 
containing 10 .mu.M pyridoxal phosphate and 0.01% .beta.-mercaptoethanol. 
Flow Rate: 5 ml/min. 
Sample: About 100-200 g of total protein (10-20 mg/ml) are applied on the 
first column. 
Gradient: 1! Pre-wash with solution A approximately 10 volumes until the 
OD.sub.280 drops below 0.1. 
2! Gradient: Solution B from 20%-100%. 
Fractions: Elution fractions of 200 ml are collected. The fractions 
containing rMETase are identified by activity assay and pooled. 
The second column: DEAE Sepharose FF 
Column: XK 50/30, Height: 25 cm, Volume: 500 ml 
Solution: A! 100 mM potassium chloride, 10 mM potassium phosphate (pH8.3) 
containing 10 .mu.M pyridoxal phosphate and 0.01% .beta.-mercaptoethanol. 
B! 200 mM potassium chloride, 10 mM potassium phosphate (pH8.3) 
containing 10 .mu.M pyridoxal phosphate and 0.01% .beta.-mercaptoethanol. 
Flow Rate: 5 ml/min. 
Sample: Approximately 10-20 g of total protein (2-4 mg/ml), after dialysis 
in 100 mM potassium chloride, 10 mM potassium phosphate (pH8.3) containing 
10 .mu.M pyridoxal phosphate for 24 hours, are applied on the second 
column. 
Gradient: 1! Pre-wash with solution A approximately 5 volumes until the 
OD.sub.280 drops below 0.05. 
2! Gradient: Solution B from 0%-60%. 
Fractions: Elution fractions of 200 ml are collected. The fractions 
containing rMETase are identified by the activity assay and pooled. 
The third column: Sephacryl S-200 HR 
Column: HiPrep 26/60, volume 320 ml. 
Solution: 0.15M sodium chloride in 10 mM sodium phosphate (pH7.2) 
Flow Rate: 1.2 ml/min. 
Sample: Approximately 10 ml concentrated sample. (after dialysis in 0.15M 
sodium chloride, 10 mM sodium phosphate (pH7.2) for 12 hours), are applied 
to the third column. 
Fractions: Elution fractions of 20 ml containing rMETase, which are 
identified by yellow color and activity assay, are collected. 
The fourth column: Acticlean.RTM. Etox 
Purified rMETase (10-20 mg protein/ml) in a volume of 100-200 ml is applied 
on a 500 ml Acticlean.RTM. Etox column, and eluted with elution buffer 
(0.15M sodium chloride in 10 mM sodium phosphate pH7.2) in order to 
eliminate endotoxin. Acticlean.RTM. Etox is reusable and can be cleaned 
with 1M sodium hydroxide and can be autoclaved. 
Concentration of the final eluant 
The final eluant is concentrate with 30K Amicon Centriprep Concentrators. 
The formulation for purified rMETase is 0.15M sodium chloride, 10 mM 
sodium phosphate, pH7.2. 
Purification of rMETase.Histidine: Chromatography on Ni.sup.++ Sepharose 
column 
The cell homogenate, after pre-column treatment, is suspended in binding 
buffer (5 mM imidazole, 0.5M NaCl, 20 mM Tris.HCL, pH7.9). The column is 
then washed with 10 volumes of binding buffer followed by washes with 6 
volumes of wash buffer (60 mM imidazole, 0.5M sodium chloride, 20 mM Tris, 
HCl, pH7.9). Elution occurs after 6 volumes of elution buffer (1M 
imidazole, 0.5M NaCl, 20 mM Tris. HCl pH7.9) have been run through the 
column. The fractions containing rMETase, identified by yellow color, are 
collected. 
EXAMPLE 5 
Analysis for The Purity of rMETase with HPLC 
Column: SUPELCO, 8-08541, Progel TM-TSK, G 3000-SWXL, 30 cm.times.7.8 mm. 
Eluent Solution: 0.15M sodium chloride in 10 mM sodium phosphate buffer 
(pH7.2). 
Flow Rate: 1 ml/min. 
Sample: 20 .mu.l (0.1-1 mg/ml). 
An example for production of rMETase is shown in FIGS. 2 and 3. Purity is 
shown in FIG. 4. 
EXAMPLE 6 
Formulations Containing Recombinant Methioninase, Crystallized and 
Lyophilized Forms 
Solution formulation: 
rMETase is formulated in solution, 0.15M sodium chloride, 10 mM sodium 
phosphate buffer (pH 7.2), at the concentration 10-20 mg/ml. The stability 
of rMETase is showed in FIG. 5. 
Crystallized form: 
rMETase (10-20 mg/ml), in a 0.15M sodium chloride and 10 mM sodium 
phosphate buffer (pH 7.2) was desalted using a Sephadex G-25 (DNA grade, 
superfine, Sigma) column. The solution was frozen on a dry ice and acetone 
bath and then crystallized in a vacuum of 100 milifar, at -80.degree. C., 
for 72 hours using a Verdis Freeze Mobil 24. 
Lyophilized form: 
rMETase (10-20 mg/ml), in a 0.15M sodium chloride and 10 mM sodium 
phosphate buffer (pH 7.2), was frozen on a dry ice and acetone bath and 
lyophilized in a vacuum of 100 milifar, at -80.degree. C., for 72 hours 
using a Verdis Freeze Mobil 24. 
Assay for activity: 
The assay was carried out in a 1 ml volume of 50 mM phosphate buffer pH 
8.0, containing 10 .mu.M pyridoxal phosphate and 10 mM methionine for 10 
min. at 37.degree. C. with varying amounts of enzyme. The reaction was 
stopped by adding 0.5 ml of 4.5% TCA. The suspension was centrifuged at 
15K rpm for 2 min. 0.5 ml of supernatant with 0.5 ml of 0.05% 
3-methyl-2-benzothiazolinone hydrazone in 1 ml of 1M sodium acetate pH 5.2 
was incubated at 50.degree. C. for 30 min. And .alpha.-Ketobutyrate was 
then determined by spectrophotometry at OD.sub.335. The amount of protein 
was determined by the procedure of Lowry Reagent Kit (Sigma). The specific 
activity was calculated as units/mg protein. 
The activity of rMETase were compared, and the results showed no big 
difference between different formulations. 
EXAMPLE 7 
Chemical Modification of Recombinant Methioninase 
The purified rMETase was formulated in a 0.15M sodium chloride in 10 mM 
sodium phosphate buffer (pH 7.2) at a concentration between 0.1M and 0.2M. 
The activity was approximately 20 units/mg. 
M-SC 5000 PEG molecular weight 5000 (Methoxy-SC-PEG, MW 5000 from 
Shearwater polymers Inc.), was dissolved in 20 mM sodium phosphate buffer 
(pH 8.3) at a concentration between 2 mM and 20 mM. The molar rations of 
M-SC 5000 PEG to rMETase are varied from 10:1 to 120:1. 
The PEGylation reactions were carried out in reaction buffer (25 mM sodium 
phosphate buffer, pH 8.3), at 20.degree. C. for 60 minutes. The reactions 
were stopped with stop buffer (0.14M sodium phosphate buffer, pH 6.5) at 
0.degree. C. Unreacted M-SC 5000 PEG was then removed with 30K Amicon 
Centriprep Concentrators. The resulting PEG-methioninase was formulated in 
0.15M sodium chloride and 10 mM sodium phosphate (pH 7.2) while 
centrifuging with 30k Amicon Centriprep concentrators. 
Analysis of PEG-rMETase in vitro 
PEG-rMETase were analyzed by activity assay, electrophoresis and HPLC, 
FIGS. 6-8. 
Activity Assay 
The activity of PEG-rMETase were between 80% to 20% of the unmodified 
rMETase. 
Electrophoresis 
PEG-rMETase were applied by both native and SDS-PAGE. 
HPLC analysis: 
PEG-rMETase were applied to a gel filtration column, no original rMETase 
peak was detected, only the PEG-METase peak were observed. The retention 
time (RT) were shorter along with the molecular ratios of PEG and rMETase 
increased. 
Pharmacokinetics of PEG-rMETase: 
Purified endotoxin-free PEG-rMETase were injected into the tail-vein of 
mice. The blood samples were collected every two hours. The levels of 
rMETase were measured by activity assay (FIG. 8). 
EXAMPLE 8 
Efficacy and Toxicity of Recombinant Methioninase 
1. Growth Inhibition of KB3-1 Cells by rMETase in vitro 
KB3-1 cells (Human squamous cell carcinoma) were grown in RPMI 1640 medium 
supplemented with 10% FBS. Various concentrations of rMETase were added to 
the medium and incubated at 37.degree. C., 5% CO.sub.2. The relative cell 
number was measured at OD.sub.570. The results demonstrated that rMETase 
effectively inhibited cell growth (FIG. 9). 
2. Growth Inhibition of KB3-1 Cells by rMETase in Nude mice 
2.times.10.sup.5 cells were injected into Balb/c nu/nu, female, mice in 
groups of eight. Control: normal saline. Group I: rMETase 30 units, Group 
II: rMETase 100 units; ip twice a day from day 5 to day 14. The tumor size 
and body weight were measured. The blood was collected on day 18. The 
results demonstrated that rMETase effectively inhibited tumor growth 
without loss of body weight and effected on blood cell production (FIGS. 
10-14). 
3. Pilot Phase I Clinical Trial of purified, natural METase 
A pilot Phase I clinical trial has been initiated in order to determine 
methioninase toxicity, pharmacokinetics of methioninase and 
methionine-depletion and maximum tolerated dose. A two hour i.v. infusion 
of 5,000 units (0.4 g) and 10,000 units (0.8 g) and a ten hour i.v. 
infusion of 20,000 units (1.6 g) of methioninase has been administered 
into patient-1, patient-2, and patient-3, respectively. All patients had 
advanced breast cancer. Blood and urine samples were obtained at frequent 
intervals from 0 to 24 hours. The toxicity evaluations were carried out 
according to WHO criteria. Pharmacokinetics data were obtained for both 
methioninase and methionine levels in the serum, FIGS. 15-18. No acute 
clinical toxicity was observed whatsoever with all toxicity criteria 
measured in patient-1, patient-2 and patient-3. The depletion of serum 
methionine started within 30 min. of the infusion, and was maintained for 
4 hours after the infusion was completed in patient-1 and patient-2. The 
lowest serum methionine levels were 35% and 19% of the pretreatment level, 
respectively, in patient-1 and patient-2 . Patient-3 who received a ten 
hour i.v. infusion of 20,000 units of recombinant methioninase without any 
signs of side effects maintained serum levels of recombinant methioninase 
as high as 50% of the maximum level for a subsequent 10 hours after 
infusion. Methionine was depleted over 200-fold from 23.1 .mu.M to 0.1 
.mu.M to 10 hours of infusion. No clinical toxicity was observed 
whatsoever in all the toxicity criteria measured in patient-3. The results 
of recombinant methioninase pilot Phase I clinical trial suggested that 
i.v. infusion of recombinant methioninase is safe and effectively depletes 
serum methionine without any signs of side effects. Clinical studies are 
continuing to determine the maximum length of time essentially complete 
serum methionine depletion can be tolerated in order to proceed to 
efficacy studies. 
4. Pilot Phase I Clinical Trial of purified, recombinant METase 
Patient 1, female, 50 years old, with stage IV breast carcinoma with lymph 
nodes metastasis, received 20,000 units (0.5 g) rMETase iv infusion for 10 
hours. Physical examinations were recorded and blood samples were 
collected before treatment, during treatment every two hours and two hours 
and 16 hours after treatment. Laboratory determination were carried 
according to the WHO criteria. The results showed that the rMETase level 
was enhanced immediately after the start of the infusion, reached the 
highest point after 10 hours. Eight hours after the infusion was stopped 
the level was 50% of the peak and still maintained 20% of the peak 16 
hours after the infusion. The results of the laboratory examination were 
evaluated according to the WHO criteria showed no acute toxicity. FIG. 19. 
Patient-2, 48 years old, female, with state IV breast carcinoma with lymph 
nodes metastasis, received 5,000 units (0.25 g) rMETase by in infusion for 
24 hours. Patient-3, 56 years old, female, with stage III renal carcinoma, 
received 10,000 units (0.5 g) rMETase by iv infusion for 24 hours. 
Physical examinations were recorded and the blood samples were collected 
before treatment, during treatment every two hours and two hours and 48 
hours after infusion. Laboratory determinations were carried out according 
to WHO criteria. The results showed that the rMETase levels were enhanced 
immediately after the start of the infusion and maintained high level 
during the infusion. After 48 hours, the methioninase level was dropped 
back normal. 
The serum methionine levels are currently being analyzed. 
The results of the laboratory examinations were evaluated according to WHO 
criteria and showed no acute toxicity (Tables 2 and 3). 
The result suggested that rMETase did not cause any toxicity in patient-2 
and patient-3. 
TABLE 2 
______________________________________ 
PROTOCOL OF rMETase 
CLINICAL PHASE I TRIAL 
Patient I Patient II 
Patient III 
______________________________________ 
Diagnosis Breast Cancer with metastasis 
Renal cancer 
Sex Female Female Female 
Age 50 48 56 
Methioninase 
10,000 units 5000 units 
10,000 units 
i.v. infusion 
8 hours 24 hours 24 hours 
Blood collection 
Before infusion and during infusion 
every two hours, After infusion 48 hours 
Evaluation 
WHO Criteria 
______________________________________ 
AntiCancer Inc. 
TABLE 3 
______________________________________ 
TOXICITY OF rMETase 
PILOT-CLINICAL PHASE I TRIAL) 
Physical & Laboratory 
Grade 
Examination Patient 1 Patient 2 
Patient 3 
______________________________________ 
Hematological 0 0 0 
Gastrointestinal 
0 0 0 
Renal 0 0 0 
Pulmonary 0 0 0 
Fever 0 0 0 
Allergic 0 0 0 
Phlebitis 0 0 0 
Cutaneous 0 0 0 
Cardiac 0 0 0 
Neurological 0 0 0 
______________________________________ 
*According to WHO toxicity criteria 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 8 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1369 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 48..1241 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GCCGGTCTGTGGAATAAGCTTATAACAAACCACAAGAGGCGGTTGCCATGCACGGC56 
MetHisGly 
TCCAACAAGCTCCCAGGATTTGCCACCCGCGCCATTCACCATGGCTAC104 
SerAsnLysLeuProGlyPheAlaThrArgAlaIleHisHisGlyTyr 
51015 
GACCCCCAGGACCACGGCGGCGCACTGGTGCCACCGGTCTACCAGACC152 
AspProGlnAspHisGlyGlyAlaLeuValProProValTyrGlnThr 
20253035 
GCGACGTTCACCTTCCCCACCGTGGAATACGGCGCTGCGTGCTTTGCC200 
AlaThrPheThrPheProThrValGluTyrGlyAlaAlaCysPheAla 
404550 
GGCGAGCAGGCCGGCCATTTCTACAGCCGCATCTCCAACCCCACCCTC248 
GlyGluGlnAlaGlyHisPheTyrSerArgIleSerAsnProThrLeu 
556065 
AACCTGCTGGAAGCACGCATGGCCTCGCTGGAAGGCGGCGAGGCCGGG296 
AsnLeuLeuGluAlaArgMetAlaSerLeuGluGlyGlyGluAlaGly 
707580 
CTGGCGCTGGCCTCGGGCATGGGGGCGATCACGTCCACGCTATGGACA344 
LeuAlaLeuAlaSerGlyMetGlyAlaIleThrSerThrLeuTrpThr 
859095 
CTGCTGCGCCCCGGTGACGAGGTGCTGCTGGGCAACACCCTGTACGGC392 
LeuLeuArgProGlyAspGluValLeuLeuGlyAsnThrLeuTyrGly 
100105110115 
TGCACCTTTGCCTTCCTGCACCACGGCATCGGCGAGTTCGGGGTCAAG440 
CysThrPheAlaPheLeuHisHisGlyIleGlyGluPheGlyValLys 
120125130 
CTGCGCCATGTGGACATGGCCGACCTGCAGGCACTGGAGGCGGCCATG488 
LeuArgHisValAspMetAlaAspLeuGlnAlaLeuGluAlaAlaMet 
135140145 
ACGCCGGCCACCCGGGTGATCTATTTCGAGTCGCCGGCCAACCCCAAC536 
ThrProAlaThrArgValIleTyrPheGluSerProAlaAsnProAsn 
150155160 
ATGCACATGGCCGATATCGCCGGCGTGGCGAAGATTGCACGCAAGCAC584 
MetHisMetAlaAspIleAlaGlyValAlaLysIleAlaArgLysHis 
165170175 
GGCGCGACCGTGGTGGTCGACAACACCTACTGCACGCCGTACCTGCAA632 
GlyAlaThrValValValAspAsnThrTyrCysThrProTyrLeuGln 
180185190195 
CGGCCACTGGAGCTGGGCGCCGACCTGGTGGTGCATTCGGCCACCAAG680 
ArgProLeuGluLeuGlyAlaAspLeuValValHisSerAlaThrLys 
200205210 
TACCTGAGCGGCCATGGCGACATCACTGCTGGCATTGTGGTGGGCAGC728 
TyrLeuSerGlyHisGlyAspIleThrAlaGlyIleValValGlySer 
215220225 
CAGGCACTGGTGGACCGTATACGTCTGCAGGGCCTCAAGGACATGACC776 
GlnAlaLeuValAspArgIleArgLeuGlnGlyLeuLysAspMetThr 
230235240 
GGTGCGGTGCTCTCGCCCCATGACGCCGCACTGTTGATGCGCGGCATC824 
GlyAlaValLeuSerProHisAspAlaAlaLeuLeuMetArgGlyIle 
245250255 
AAGACCCTCAACCTGCGCATGGACCGCCACTGCGCCAACGCTCAGGTG872 
LysThrLeuAsnLeuArgMetAspArgHisCysAlaAsnAlaGlnVal 
260265270275 
CTGGCCGAGTTCCTCGCCCGGCAGCCGCAGGTGGAGCTGATCCATTAC920 
LeuAlaGluPheLeuAlaArgGlnProGlnValGluLeuIleHisTyr 
280285290 
CCGGGCCTGGCGAGCTTCCCGCAGTACACCCTGGCCCGCCAGCAGATG968 
ProGlyLeuAlaSerPheProGlnTyrThrLeuAlaArgGlnGlnMet 
295300305 
AGCCAGCCGGGCGGCATGATCGCCTTCGAACTCAAGGGCGGCATCGGT1016 
SerGlnProGlyGlyMetIleAlaPheGluLeuLysGlyGlyIleGly 
310315320 
GCCGGGCGGCGGTTCATGAACGCCCTGCAACTGTTCAGCCGCGCGGTG1064 
AlaGlyArgArgPheMetAsnAlaLeuGlnLeuPheSerArgAlaVal 
325330335 
AGCCTGGGCGATGCCGAGTCGCTGGCGCAGCACCCGGCAAGCATGACT1112 
SerLeuGlyAspAlaGluSerLeuAlaGlnHisProAlaSerMetThr 
340345350355 
CATTCCAGCTATACCCCAGAGGAGCGTGCGCATTACGGCATCTCCGAG1160 
HisSerSerTyrThrProGluGluArgAlaHisTyrGlyIleSerGlu 
360365370 
GGGCTGGTGCGGTTGTCGGTGGGGCTGGAAGACATCGACGACCTGCTG1208 
GlyLeuValArgLeuSerValGlyLeuGluAspIleAspAspLeuLeu 
375380385 
GCCGATGTGCAACAGGCACTCAAGGCGAGTGCCTGAACCCGTCACGGATGAGG1261 
AlaAspValGlnGlnAlaLeuLysAlaSerAla 
390395 
TCAATGCAATGGTGGCAATGATGAACCTTGTGCCTGGCGACGGCGTGCCCGGTGACAGCG1321 
ACCCTGGCGAAACTGCAGAGTGGCTGGAGGCGCTGGAGTCGACCCTGG1369 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 398 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetHisGlySerAsnLysLeuProGlyPheAlaThrArgAlaIleHis 
151015 
HisGlyTyrAspProGlnAspHisGlyGlyAlaLeuValProProVal 
202530 
TyrGlnThrAlaThrPheThrPheProThrValGluTyrGlyAlaAla 
354045 
CysPheAlaGlyGluGlnAlaGlyHisPheTyrSerArgIleSerAsn 
505560 
ProThrLeuAsnLeuLeuGluAlaArgMetAlaSerLeuGluGlyGly 
65707580 
GluAlaGlyLeuAlaLeuAlaSerGlyMetGlyAlaIleThrSerThr 
859095 
LeuTrpThrLeuLeuArgProGlyAspGluValLeuLeuGlyAsnThr 
100105110 
LeuTyrGlyCysThrPheAlaPheLeuHisHisGlyIleGlyGluPhe 
115120125 
GlyValLysLeuArgHisValAspMetAlaAspLeuGlnAlaLeuGlu 
130135140 
AlaAlaMetThrProAlaThrArgValIleTyrPheGluSerProAla 
145150155160 
AsnProAsnMetHisMetAlaAspIleAlaGlyValAlaLysIleAla 
165170175 
ArgLysHisGlyAlaThrValValValAspAsnThrTyrCysThrPro 
180185190 
TyrLeuGlnArgProLeuGluLeuGlyAlaAspLeuValValHisSer 
195200205 
AlaThrLysTyrLeuSerGlyHisGlyAspIleThrAlaGlyIleVal 
210215220 
ValGlySerGlnAlaLeuValAspArgIleArgLeuGlnGlyLeuLys 
225230235240 
AspMetThrGlyAlaValLeuSerProHisAspAlaAlaLeuLeuMet 
245250255 
ArgGlyIleLysThrLeuAsnLeuArgMetAspArgHisCysAlaAsn 
260265270 
AlaGlnValLeuAlaGluPheLeuAlaArgGlnProGlnValGluLeu 
275280285 
IleHisTyrProGlyLeuAlaSerPheProGlnTyrThrLeuAlaArg 
290295300 
GlnGlnMetSerGlnProGlyGlyMetIleAlaPheGluLeuLysGly 
305310315320 
GlyIleGlyAlaGlyArgArgPheMetAsnAlaLeuGlnLeuPheSer 
325330335 
ArgAlaValSerLeuGlyAspAlaGluSerLeuAlaGlnHisProAla 
340345350 
SerMetThrHisSerSerTyrThrProGluGluArgAlaHisTyrGly 
355360365 
IleSerGluGlyLeuValArgLeuSerValGlyLeuGluAspIleAsp 
370375380 
AspLeuLeuAlaAspValGlnGlnAlaLeuLysAlaSerAla 
385390395 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1369 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CCAGGGTCGACTCCAGCGCCTCCAGCCACTCTGCAGTTTCGCCAGGGTCGCTGTCACCGG60 
GCACGCCGTCGCCAGGCACAAGGTTCATCATTGCCACCATTGCATTGACCTCATCCGTGA120 
CGGGTTCAGGCACTCGCCTTGAGTGCCTGTTGCACATCGGCCAGCAGGTCGTCGATGTCT180 
TCCAGCCCCACCGACAACCGCACCAGCCCCTCGGAGATGCCGTAATGCGCACGCTCCTCT240 
GGGGTATAGCTGGAATGAGTCATGCTTGCCGGGTGCTGCGCCAGCGACTCGGCATCGCCC300 
AGGCTCACCGCGCGGCTGAACAGTTGCAGGGCGTTCATGAACCGCCGCCCGGCACCGATG360 
CCGCCCTTGAGTTCGAAGGCGATCATGCCGCCCGGCTGGCTCATCTGCTGGCGGGCCAGG420 
GTGTACTGCGGGAAGCTCGCCAGGCCCGGGTAATGGATCAGCTCCACCTGCGGCTGCCGG480 
GCGAGGAACTCGGCCAGCACCTGAGCGTTGGCGCAGTGGCGGTCCATGCGCAGGTTGAGG540 
GTCTTGATGCCGCGCATCAACAGTGCGGCGTCATGGGGCGAGAGCACCGCACCGGTCATG600 
TCCTTGAGGCCCTGCAGACGTATACGGTCCACCAGTGCCTGGCTGCCCACCACAATGCCA660 
GCAGTGATGTCGCCATGGCCGCTCAGGTACTTGGTGGCCGAATGCACCACCAGGTCGGCG720 
CCCAGCTCCAGTGGCCGTTGCAGGTACGGCGTGCAGTAGGTGTTGTCGACCACCACGGTC780 
GCGCCGTGCTTGCGTGCAATCTTCGCCACGCCGGCGATATCGGCCATGTGCATGTTGGGG840 
TTGGCCGGCGACTCGAAATAGATCACCCGGGTGGCCGGCGTCATGGCCGCCTCCAGTGCC900 
TGCAGGTCGGCCATGTCCACATGGCGCAGCTTGACCCCGAACTCGCCGATGCCGTGGTGC960 
AGGAAGGCAAAGGTGCAGCCGTACAGGGTGTTGCCCAGCAGCACCTCGTCACCGGGGCGC1020 
AGCAGTGTCCATAGCGTGGACGTGATCGCCCCCATGCCCGAGGCCAGCGCCAGCCCGGCC1080 
TCGCCGCCTTCCAGCGAGGCCATGCGTGCTTCCAGCAGGTTGAGGGTGGGGTTGGAGATG1140 
CGGCTGTAGAAATGGCCGGCCTGCTCGCCGGCAAAGCACGCAGCGCCGTATTCCACGGTG1200 
GGGAAGGTGAACGTCGCGGTCTGGTAGACCGGTGGCACCAGTGCGCCGCCGTGGTCCTGG1260 
GGGTCGTAGCCATGGTGAATGGCGCGGGTGGCAAATCCTGGGAGCTTGTTGGAGCCGTGC1320 
ATGGCAACCGCCTCTTGTGGTTTGTTATAAGCTTATTCCACAGACCGGC1369 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GCCGGTCTGTGGAATAAGCT20 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CCAGGGTCGACTCCAGCGCC20 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GGAATTCCATATGCACGGCTCCAACAAGC29 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
AGTCATCCTAGGTCACATCATCATCATCATCATGGCACTCGCCTTGAGTGC51 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
AGTCATCCTAGGTCAGGCACTCGCCTTGAGTGC33 
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