Materials and methods for determining ob protein in a biological sample

Methods for determining ob protein in a biological sample are disclosed. In a preferred embodiment, the biological samples are analyzed using an ELISA capable of detecting and quantitating ob protein.

BACKGROUND OF THE INVENTION 
Obesity affects an ever-increasing proportion of the population of Western 
cultures. Nearly one-third of adults in the United States are in excess of 
their ideal body weight by at least 20%. This results in a major public 
health problem, because obesity is associated with a multitude of medical 
problems that include hypertension, elevated blood lipids, coronary artery 
disease, osteoarthritis and Type II or non-insulin-dependent diabete 
mellitus (NIDDM). In the United States alone, there are an estimated 6-10 
million individuals with NIDDM, including 18% of the population over 65 
years of age and most of these individuals are obese (Harris et al. 
Diabetes 36:523-534, 1987). While there appears to be a heterogeneous 
etiology for NIDDM, obesity alone leads to insulin resistance and NIDDM in 
individuals that are predisposed to the disease, and it exacerbates the 
condition in patients already presenting with NIDDM. 
Stability of body composition requires that energy intake equals 
expenditure when integrated over prolonged periods. Since recent human 
studies have failed to demonstrate active changes in energy expenditure 
with changes in body composition, it appears likely that energy intake is 
continually adjusted to preserve a constant total adipose tissue mass. If 
adipose tissue mass is regulated directly, then there must be some input 
signaling this quantity to the central nervous system for the purpose of 
making corrective changes in appetite when total body fat content 
fluctuates. The nature of this input has been examined in a variety of 
animal experiments involving induced weight change (Cohn et al., Yale J. 
Biol. Med. 34:598-607, 1962; Harris et al., Proc. Soc. Exp. Biol. Med. 
191:82-89, 1989; and Wilson et al., Am. J. Physiol. 259:R1148-R1155, 
1990); lipectomy (Forger et al., Metabolism 37:782-86, 1988; Liebelt et 
al., Ann. N.Y. Acad. Sci. 131:559-82, 1965; and Chlouverakis et al., 
Metabolism 23:133-37, 1974); plasma transfer from obese or satiated 
animals to hungry animals (Davis et al., Science 156:1247-48, 1967; Davis 
et al., J. Comp. Physiol. Psychol. 67:407-14, 1969; and King, Physiol. 
Psychol. 4:405-08, 1976); and parabiosis between obese and lean animals 
(Hervey, J. Physiol. 145:336-52, 1959; Parameswaran et al., Am. J. 
Physiol. 232:R150-R157, 1977; Nishizawa et al., Am. J. Physiol. 
239:R344-351, 1980; Harris et al., Am. J. Physiol. 257:R326-R336, 1989; 
Schmidt et al., Acta Physiol. Acad. Sci. Hung. Tomus 36:293-98, 1969; 
Coleman et al., Diabetologia 9:294-98, 1973; Harris et al., Int. J. 
Obesity 11:275-83, 1987; and Coleman et al., Am. J. Physiol. 
217:1298-1304, 1969). From these experiments, there was some evidence that 
the plasma level of one or more unidentified stable circulating molecules 
increases in proportion to total body fat content and augments the effect 
of meal-related satiety signals in the central nervous system. 
The search for factors that affect and ultimately control appetite has 
taken two paths. The first, based on genetic observations, has recently 
resulted in the cloning of the ob gene (Zhang et al., Nature 372:425-32, 
1994) by use of positional cloning using mice that were ob/ob mutants. 
However, while Zhang and others speculated that the ob gene product may be 
a regulating factor of body fat content, this has not been shown. The 
second path involves attempts to isolate a discrete factor or factors that 
regulate body composition based on functional properties. 
The isolation of a functional appetite suppression factor (ASF), however, 
has been unsuccessful, in part due to an inability to reliably determine 
changes in food consumption as a result of administration of putative 
appetite suppression factors. The present invention provides a consistent 
method for identifying and quantitating ASF activity in a test sample, 
including a sample containing an expressed ob gene product or other 
factors that regulate appetite. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a method for 
determining ob protein in a biological sample, comprising combining a 
biological sample and a capture antibody capable of binding ob protein, 
wherein the capture antibody is immobilized on a solid matrix, thereby 
forming a captured ob-solid matrix; removing unbound molecules from the 
captured ob-solid matrix; incubating a detecting antibody capable of 
binding ob protein and capable of generating a detectable signal with the 
captured ob-solid matrix, thereby forming a detection complex; removing 
unbound detecting antibody from the detection complex; and determining the 
detectable signal generated by the detection complex. In another 
embodiment, the method further comprises, after the step of determining, 
the step of comparing the detectable signal generated by the detection 
complex associated with the biological sample with a standard curve of ob 
protein samples.

DETAILED DESCRIPTION OF THE INVENTION 
Prior to describing the present invention in detail, it may be helpful to 
define certain terms used herein: 
ob/ob mice: Inbred mice that are homozygous for an inactivating mutation at 
the ob (obese) locus. ob/ob mice are hyperphagic and hypometabolic, and 
are believed to be deficient in production of circulating satiety factor. 
db/db mice: Inbred mice that are homozygous for an inactivating mutation at 
the db (diabetes) locus. db/db mice display a phenotype similar to that of 
ob/ob mice, except db/db mice also display a diabetic phenotype. db/db 
mice are believed to be resistant to the effects of circulating satiety 
factor. 
fa/fa rats: Also known as Zucker rats, fa/fa rats are homozygous for a 
mutation that is syntenic to the mutation in db/db mice. However, fa/fa 
rats (and particularly young fa/fa rats) are not as severely diabetic as 
db/db mice, and thus exhibit a phenotype and characteristics that are 
similar to ob/ob mice. 
Hyperphagia: Ingestion of greater than optimal quantity of food. 
Microdissection: For tissue that has been excised and snipped into 
pea-sized lumps, the process of floating such lumps on the surface of a 
liquid medium and dissecting such lumps further by snipping with scissors 
held vertically to the liquid's surface. 
Adipose conditioned medium (ACM): Serum-free culture medium that has been 
incubated with microdissected, excised adipose tissue obtained from a 
mammalian donor. 
Appetite suppression factor (ASF): A factor that, upon administration of an 
effective dose to an appropriate recipient mammal, down-regulates food 
intake in that recipient. As used herein, ASF includes the terms "satiety 
factor", "adipose satiety factor" and "appetite (down-)regulating factor". 
ob: As used herein, ob or ob denotes nucleic acid. This designation is 
distinct from the rodent mutant phenotype designations defined above 
(i.e., ob/ob mice), which are used in the format "*/*", but not in the 
singular "*". 
ob: As used herein, ob denotes protein. 
One aspect of the present invention describes methods useful for 
identifying appetite regulating factors. In a preferred embodiment, the 
methods are useful for identifying factors that down-regulate appetite. 
The methods feature administration of a test sample that putatively 
contains an appetite regulating factor to a recipient mammal. In a 
preferred embodiment, the recipient mammal exhibits a relatively constant 
baseline level of chow intake prior to administration of the test sample. 
Another aspect of the claimed invention discloses a product having ASF 
activity. In a preferred embodiment, ASF is secreted by adipose tissue 
into serum-free, mammalian cell culture medium. This conditioned medium is 
fractionated using ion exchange chromatography and a 0-0.5M NaCl linear 
gradient. Eluted fractions having ASF activity are described herein. 
A further aspect of the present invention describes a method for reducing 
food intake in a mammal. Such method features administration of a 
pharmaceutical composition comprising one or more proteins that correspond 
to sequences of amino acids described in further detail herein. 
A. Methods for Identifying Factors that Regulate Appetite 
The present invention provides methods for identification of factors that 
regulate appetite. These methods can be used to isolate factors that work 
independently or in concert, and may be used to isolate novel factors that 
may enhance or diminish appetite. 
Appetite suppression factors are assayed using a selected recipient that 
consumes the same amount of chow each day, by weight, within about a 20% 
variation. It is preferred that the variation by weight of chow intake not 
vary by more than 15%, and most preferably, the variation will not exceed 
10%. Within the assay of the present invention, an animal is weighed, 
peripherally injected with a physiologically acceptable buffer (for 
example, phosphate buffered saline), and fed a pre-weighed, test amount of 
chow each day at the same time. By peripheral injection, it is meant that 
the injection is peripheral to the central nervous system. An exemplary 
peripheral injection includes, but is not limited to, those administered 
intravenously, subcutaneously, intramuscularly, intranasally, and 
intraperitoneally. In a preferred embodiment, the injection is 
administered intraperitoneally. By injecting the animal each day at the 
same time (.+-.60 minutes), the animal becomes accustomed to daily 
injection and handling procedures prior to injection of the test sample. 
After 24 hours, the remaining chow is removed and weighed. The animal is 
again weighed and test amount chow is replaced. Other factors that are 
preferably controlled include light exposure and temperature. Injections 
should be administered at the time of day that is associated with the 
onset of spontaneous feeding. In a preferred embodiment, the assay animal 
is a rodent that is preferably exposed to low levels of light (typically 
100-200 watts in the room for 12 hours daily), kept at 
21.degree..+-.2.degree. C. and fed in the dark to optimize spontaneous 
feeding. 
In one embodiment, an assay (or recipient) mammal is deficient in the 
production of a factor that decreases food consumption, but is responsive 
to exogenously administered factor. Accordingly, the recipient is highly 
sensitive to the factor if it is present in a test sample. Examples of 
mammals that are deficient in appetite suppression factors include, for 
instance, the ob/ob mouse (such as a C57BL/6J ob/ob mouse). Transgenic 
mice and mice that exhibit a complete absence of gene function, referred 
to as "knockout mice" (Snouwaert et al., Science 257:1083, 1992), may also 
be used (Lowell et al., Nature 366:740-742, 1993). However, normal animals 
have been used successfully in the methods of the present invention, and 
include, for example, BALB/c mice and KSJ mice. It is preferred that the 
recipient animals be male. 
Once it has been established that an animal's daily chow intake does not 
vary more than 20%, the animal is weighed, injected with: (1) a test 
sample; (2) a control buffer used in the preparation of the test sample; 
or (3) PBS, and presented the test amount of chow. Daily monitoring is 
done by weighing the animal. After completion of the testing period, the 
animal is returned to receiving a daily PBS injection and chow consumed is 
measured, until the animal's daily chow consumption has returned to its 
baseline. An injection of test sample, buffer or PBS may be given once a 
day, or multiple injections may be administered to examine prolonged 
effects on food consumption. The number of injections will be limited by 
the test animal's tolerance and ability to achieve a baseline feeding 
profile, as described above. A test sample may be administered once or 
sequentially over a period of one or more days. If it is desired to 
measure weight loss, in addition to a decrease in food consumption, then 
sequential, multiple day injections will be preferred. In a preferred 
embodiment, an injection is administered once or twice during one 24 h 
period. 
Changes in metabolic factors that regulate appetite and obesity can be 
measured. For example, changes in glucose and insulin levels can be 
determined. Blood samples collected at discrete intervals before, during 
and after administration of a putative ASF are analyzed for changes in the 
metabolic factors. 
Test samples contain a putative appetite suppression factor or factors to 
be tested in a physiologically acceptable solution. Examples of 
physiologically acceptable solutions include, but are not limited to, PBS 
and protein-free cell culture medium that minimally contains a carbon 
source, a nitrogen source, essential amino acids, vitamins and minerals. 
Selection of buffers that are physiologically acceptable are well known in 
the art (see, for example, Remington's Pharmaceutical Science, 16th ed., 
Mack Publishing Company, Easton, Pa. (1982)), which is incorporated herein 
by reference. Determination of acceptable doses of total protein 
containing a putative appetite suppression factor is well within the skill 
of one ordinarily skilled in the art. For example, in mice, doses of 
conditioned medium that has appetite suppression activity generally are in 
the range of 0.25 to 1.75 mg protein/g recipient, with preferred doses in 
a range of 0.5 to 0.75 mg protein/g recipient. One skilled in the art 
would recognize that, upon purification of a product having appetite 
suppression activity, the total protein concentration per dose would 
decrease. In mice, for instance, doses of purified ob protein generally 
are in the range of 0.01 to 4 .mu.g protein/g recipient/day, with 
preferred doses in a range of 0.1 to 0.5 .mu.g protein/g recipient/day. 
Determination of a decrease in food consumption by the test animal is made 
by comparing: (1) chow consumption during a 24 hour test period following 
the time when the animal was injected with the test sample or buffer to 
(2) chow intake during each 24 hour period preceding, when PBS alone was 
injected. Additional comparisons between animals given test samples and 
control animals are performed. The chow intake during the test period may 
be expressed as a percentage of the mean chow intake from the pre- and 
post-testing periods (i.e., baseline intake). A significant decrease in 
food consumption is a reduction to at least 85% of baseline (BL) intake, 
and preferably at least 75% of baseline. 
An in vitro assay to detect an ASF is also advantageous. Preferably, such 
in vitro assay enables detection and quantitation of biologically active 
ASF. An in vitro assay can also be used to evaluate changes in metabolic 
factors regulating appetite and obesity in response to the in vivo 
administration of a putative ASF. In one embodiment, the in vitro assay is 
cell-based, and involves either primary or immortalized cultured mammalian 
cells. Exemplary mammalian cells in this regard include, but are not 
limited to, islet cells (beta, delta or alpha), adrenal chromaffin cells, 
neuronal lineage cells, adipocytes and hypothalamic cells. With an islet 
cell or adrenal chromaffin cell assay, regulation of insulin secretion by 
a putative ASF can be examined. Alternatively, modulation of triglyceride 
production, or of glucocorticoid and/or catecholamine production, can be 
assayed. With neuronal lineage cells, a putative ASF can be tested for 
hormone-sensitive ion modulation (for instance, dopaminergic and/or 
serotoninergic effects). With adipocytes, a putative ASF can be examined 
for autocrine/paracrine effects on the adipocytes, such as 3T3-L1 cells. 
With hypothalamic cells, binding of a putative ASF to histologic sections 
of hypothalamus may be determined. Any mammalian cell or cell line that 
enables detection and quantitation of biologically active ASF may be used 
to identify a cell line(s) that is an appropriate source for creation of 
an ASF receptor cDNA library. 
B. Appetite Suppression Factor 
Microdissected adipose tissue fragments were obtained from db/db mice or, 
preferably, fa/fa rats. As demonstrated in parabiosis studies, both fa/fa 
rats and db/db mice are insensitive to a circulating factor(s) that 
reduces food intake. A current hypothesis proposes that these mice and 
rats have a defective receptor that is incapable of recognizing this 
circulating factor. Yet a further hypothesis proposes that these mice and 
rats produce elevated levels of circulating factor, due to the absence of 
a functioning receptor and complete feedback loop. 
Because adipose tissue excised from fa/fa rats is approximately 10 times 
greater in mass than that obtained from db/db mice, fa/fa rat adipose 
tissue is preferred for production of adipose conditioned medium (ACM). 
The present invention discloses that appetite suppression factor (ASF) is 
synthesized and secreted by adipose tissue. When excised, microdissected 
and minced adipose tissue was cultured short term in serum-free tissue 
culture medium, the resultant ACM contained one or more factors that 
suppressed food consumption in recipient mice. When adipose tissue was 
cultured in the presence of .sup.35 S-methionine, incorporation of .sup.35 
S label into trichloroacetic acid (TCA)-insoluble material in the ACM was 
linear over culturing periods up to 24 h. This incorporation of .sup.35 S 
label into acid-insoluble material was blocked by incubation of adipose 
tissue in 0.2 mg/ml cycloheximide. These results are consistent with 
adipose tissue being more than a mere repository for ASF that has been 
synthesized and secreted by another tissue. In fact, these data are 
consistent with adipose tissue's ability to synthesize and secrete ASF de 
novo. 
When concentrated ACM was injected intraperitoneally into BALB/c mice 
(normal mice), it caused a significant dose-dependent suppression of 24 h 
chow intake when appropriate assay conditions were used. This appetite 
down-regulation was not observed following intraperitoneal injection of 
the following substances: (a) bovine fraction V albumin; (b) excised, 
microdissected and minced liver tissue conditioned medium; (c) serum 
obtained from male db/+ mice; (d) BAF3 24-11 cell 
(thrombopoietin-secreting) conditioned medium; and (e) human fibrinogen. 
These results are consistent with secretion and synthesis of a specific 
appetite down-regulating factor by adipose tissue. 
ACM prepared using adipose tissue obtained from various anatomic sites 
yielded comparable appetite suppression activity. More specifically, 
adipose tissue was dissected from fa/fa rat epididymal, inguinal and 
dorsal fat pads. When these adipose tissue samples were cultured 
separately, all conditioned media produced comparable reduction in food 
intake upon intraperitoneal injection. 
Approximately 80% of secreted ASF was present in ACM between 5 and 90 
minutes of incubation. After 300 minutes of culturing, an additional 20% 
increase in ASF was observed. These results were consistent with the 
appearance of .sup.35 S-labeled acid precipitable material. Moreover, 
these data provide evidence that ASF is not merely a contaminant residing 
in adipose tissue. 
ASF in ACM was stable to freezing at -70.degree. C. for 2 months; thawing 
(5 minutes at 37.degree. C.); dilution (activity was recoverable upon 
reconcentration of 1:10 dilutions of conditioned medium); and 
concentration by ultrafiltration. However, appetite down-regulating 
activity was destroyed by heating the ACM at 95.degree. for 5 min. ASF was 
present in a 66% saturated ammonium sulfate precipitate of ACM, but was 
eliminated upon exposure to 6M guanidine hydrochloride. Because 
protease-treated ACM could not be safely administered to mice, the in vivo 
effect of protease treatment of ACM was not determined. These observations 
provide evidence that the active factor(s) in ACM is a protein. 
ACM was fractionated using membrane ultrafiltration and membranes that 
retain various sizes of molecules. More specifically, adipose tissue was 
excised from 3 Zucker rats, and the resultant ACMs were pooled. The pooled 
material was concentrated using a 1 kD cut-off ultrafiltration membrane, 
and stored in 1 ml aliquots at -70.degree. C. The protein concentration in 
this concentrated ACM was calculated to be about 13 mg/ml. In the mouse 
bioassay, this preparation reduced chow intake to 44.+-.11% of baseline. 
Subsequently, five 1 ml aliquots were thawed and diluted 1:10 in PBS. The 
dilute ACM was fractionated using a Centriprep 3, 10, or 30, or a 
Centricon 100 apparatus. The ACM retentates had protein concentrations of 
5.5, 7.0, 6.4 and 6.0 mg/ml, respectively. When retentates were tested in 
the mouse bioassay, chow intake was determined to be 97, 43, 86 and 67% 
BL, respectively. 
The appetite suppression that was obtained using fractionated ACM retentate 
from the Centriprep 30 column was similar to that of the unfractionated 
ACM, which contained about twice the concentration of protein. More 
specifically, administration of equal volumes of preparations provides the 
following ratios: fractionated, concentrated ACM=44% BL/13 mg/ml protein; 
fractionated, &gt;30 kD, concentrated ACM=43% BL/7.0 mg/ml. This fractionated 
composition may also be termed "isolated", meaning that the fractionated 
composition is found in a condition other than its original environment, 
such as unconcentrated and unfractionated conditioned medium. 
Alternatively, Zucker rat ACM has been subjected to HPLC and fractionated. 
The HPLC fractions represent "isolated" preparations, also. In fact, 
HPLC-isolated compositions exhibited less complexity (i.e., more 
isolation) than ultrafiltration retentates. 
The ASF secreted into ACM was not tumor necrosis factor alpha 
(TNF-.alpha.), a known anorexic agent produced by adipose tissue. The 
concentration of TNF-.alpha. in ACM, as measured by an L929 cell 
cytotoxicity assay (Aggarwal et al., J. Biol. Chem. 260: 2345, 1985), was 
over 100-fold less than the concentration of purified recombinant mouse 
TNF-.alpha. required to suppress feeding in the assay of the present 
invention. 
To demonstrate that the ASF present in ACM is capable of acting on the 
central nervous system (CNS), adipose tissue was excised from Long-Evans 
rats (normal outbred rats) that had been gavage overfed to produce obesity 
and a complete cessation of spontaneous chow intake. When this Long-Evans 
ACM was injected intracerebrally into recipient rats, a significant 
feeding suppression was observed, as compared to recipients that were 
injected with unconditioned medium alone. The central effective dose was 
1000-fold less than the peripheral effective dose of Long-Evans ACM, 
consistent with a central locus of ASF action. 
ASF may increase CNS responsiveness to gastrointestinal meal termination 
signals, resulting in reduced average meal size or frequency. The 
hyperphagia of the ob/ob mouse may generate a high level of 
gastrointestinal feedback to the CNS, therefore ob/ob mice may be 
unusually sensitive to exogenously administered ASF. In fact, 
intraperitoneal injection of ACM resulted in greater feeding suppression 
in ob/ob mouse recipients than in BALB/c recipient mice. Moreover, the 
duration of feeding suppression in ob/ob recipients was about 3 days, as 
compared to about 1 day for BALB/c recipients. 
Because of the enhanced responsiveness of ob/ob recipients to injections of 
ACM, ob/ob recipients are preferred recipients for injection of test 
samples that putatively contain ASF. In a preferred embodiment, an ob/ob 
recipient is injected intraperitoneally with a test sample. When ACM is 
the test sample, a single 1 ml injection of medium that has been 
conditioned for 3-5 h, then concentrated 20.times. by ultrafiltration 
through a 1000 Dalton molecular weight cut-off membrane, is preferred. 
The apparent hypersensitivity of ob/ob mice to injection of ACM did not 
result from a non-specific toxic effect. When db/db mice were injected 
intraperitoneally at a dose of 0.40 mg/g, the db/db recipients' 24 h food 
intake was reduced to 89% of baseline, whereas the ob/ob recipients' 24 h 
food intake was reduced to 58% of baseline. These data are inconsistent 
with non-specific toxicity of the ACM, which would have affected ob/ob and 
db/db recipients equally. However, these results are consistent with the 
previously reported insensitivity of db/db mice to parabiotic satiety 
factor (Coleman and Hummel, Am. J. Physiol. 217: 1298, 1969; Coleman, 
Diabetologia 9:294, 1973). 
A two bottle conditioned taste aversion test, a standard paradigm for 
distinguishing physiological regulators of feeding from substances that 
reduce feeding through the induction of malaise (Spear et al., Ann. N.Y. 
Acad. Sci. 443:42, 1985), confirmed that ACM acted as a specific regulator 
of feeding. Taste aversion could not be produced in ob/ob mice by pairing 
intraperitoneal injections of ACM with the presentation of a novel flavor, 
but could be produced by pairing lithium chloride, an established nauseant 
(Nachman and Ashe, Physiol. Behav. 10:73, 1973), with a novel flavor. 
Medium conditioned with adipose tissue excised from ob/ob mice exhibited 
significantly lower appetite suppression activity than media conditioned 
with adipose tissue excised from other strains of rodents. These data are 
consistent with previously reported parabiosis results, and provide 
further evidence that ACM from donors other than ob/ob mice contains a 
specific active factor. 
A single daily injection of fa/fa rat ACM produced a sustained reduction in 
chow intake and progressive weight loss when administered to ob/ob 
recipients over the course of 10 days, as compared to ob/ob recipients 
that received injections of PBS. This weight loss was attributable 
exclusively to reduced caloric (i.e., chow) intake as shown by the 
comparable rate of weight loss in pair fed animals. These data were 
inconsistent with an increased energy expenditure as a result of 
physiological or pathophysiological effects of ACM. 
In order to further characterize ACM, a large pool of fa/fa ACM was 
prepared. When an aliquot of this large pool was concentrated by 
ultrafiltration, no appetite down-regulating activity was observed upon 
injection into ob/ob recipients. The concentrated material was applied to 
an ion exchange column and eluted with a salt gradient. The most 
significant ASF activity in the resultant partially purified fractions 
eluted at a concentration of 0.1-0.5M NaCl, with fractions eluted at 
0.1-0.25M NaCl, at 0.25-0.38M NaCl, and at 0.38-0.5M NaCl preferred. 
C. Methods for Reducing Food Consumption 
The sequence of a cDNA clone encoding a representative mouse appetite 
suppression factor (mouse ob protein) is shown in SEQ ID NO:1, and the 
corresponding amino acid sequence is shown in SEQ ID NO:2. The sequences 
of the human DNA and protein are shown in SEQ ID NO:3 and SEQ ID NO:4, 
respectively. Those skilled in the art will recognize that the sequences 
shown in SEQ ID NOS. 1, 2, 3 and 4 correspond to single alleles of the 
murine and human ob genes, and that allelic variation is expected to 
exist. Allelic variants of the DNA sequences shown in SEQ ID NO:1 and 
NO:3, including those containing silent mutations and those in which 
mutations result in amino acid sequence changes, are useful within the 
present invention, as are proteins which are allelic variants of SEQ ID 
NO:2 and NO:4. 
An exemplary allelic variant of the mouse nucleic acid and protein of SEQ 
ID NO:1 and NO:2 is shown in SEQ ID NO:5 and SEQ ID NO:6. Only the mature 
protein and its coding sequence are shown. In this variant, the Gln 
residue at position 49 of SEQ ID NO:2 is not present. 
The DNA molecule shown in SEQ ID NO:1 was cloned by polymerase chain 
reaction (PCR; see Mullis et al., U.S. Pat. No. 4,683,195; Mullis, U.S. 
Pat. No. 4,683,202) using primers designed from the sequence of the mouse 
obese gene (Zhang et al., Nature 372:425-432, 1994) and cDNA made from 
db/db mouse adipose tissue mRNA. A second round of PCR was used to add 
restriction sites at the ends of the DNA to facilitate subsequent 
manipulations. The mouse DNA was used to probe a human adipose tissue cDNA 
library (obtained from Clontech, Palo Alto, Calif.), and the DNA molecule 
shown in SEQ ID NO:3 was isolated. 
The appetite suppression factor DNA and amino acid sequences disclosed 
herein are useful tools for preparing isolated polynucleotide molecules 
encoding appetite suppression factors from other species ("species 
homologs"), in particular other mammalian species. DNA molecules, 
including complementary DNA (cDNA) and genomic DNA, can be cloned by 
conventional techniques using readily available reagents. Methods for 
using sequence information from a first species to clone a correspoding 
polynucleotide sequence from a second species are well known in the art. 
See, in general, Ausubel et al., eds., Current Protocols in Molecular 
Biology, John Wiley and Sons, Inc., New York, 1987. Suitable techniques in 
this regard include polymerase chain reaction, hybridization to labeled 
probes (see, e.g., Sambrook et al., eds., Molecular Cloning: A Laboratory 
Manual, 2nd ed., Cold Spring Harbor Laboratory, 1989) and expression 
cloning using antibody probes or soluble receptors (e.g., Young and Davis, 
Proc. Natl. Acad. Sci. USA 80:1194-1198, 1983). DNA molecules encoding ASF 
proteins are generally at least 60%, preferably at least 80%, and may be 
90-95% or more identical in sequence to SEQ ID NO: 1, SEQ ID NO:3, or 
their allelic variants. 
Analysis of the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4 
indicated that each protein included an amino-terminal signal peptide of 
21 amino acid residues. The mature proteins thus begin with amino acid 
residue 22 (Val) of SEQ ID NO:2 and SEQ ID NO:4. 
For use within the present invention, it is preferred to prepare the 
representative appetite suppression factor as a recombinant protein, that 
is by introducing DNA encoding the protein into a host cell and culturing 
the cell so that the DNA is expressed and the protein can be recovered. 
DNA molecules can be introduced into cells according to conventional 
procedures. In general, a DNA molecule encoding an appetite suppression 
factor is inserted into an expression vector, where it is operably linked 
to additional DNA segments that provide for its transcription. Such 
additional segments include promoter and terminator sequences. An 
expression vector may also include one or more origins of replication, one 
or more selectable markers, an enhancer, a polyadenylation signal, etc. 
Expression vectors are generally derived from plasmid or viral DNA, or may 
contain elements of both. The term "operably linked" indicates that the 
DNA segments are arranged so that they function in concert for their 
intended purposes, e.g., transcription initiates in the promoter and 
proceeds through the coding segment to the terminator. Methods for 
introducing DNA into prokaryotic and eukaryotic cells and culturing the 
cells are well known in the art. Suitable host cells include prokaryotic 
cells (e.g., bacteria of the genera Escherichia and Bacillus), unicellular 
microorganisms (e.g., yeasts of the genera Saccharomyces, Pichia, 
Schizosaccharomyces and Kluyveromyces), and cells from multicellular 
organisms (e.g., mammalian cells, including BHK, CHO and COS cell lines; 
insect cells; avian cells; and plant cells). See, for example, Kawasaki, 
U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; Bitter, 
U.S. Pat. No. 4,977,092; Welch et al., U.S. Pat. No. 5,037,743; Murray et 
al., U.S. Pat. No. 4,766,073; Wigler et al., Cell 14:725, 1978; Corsaro 
and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, 
Virology 52:456, 1973; Neumann et al., EMBO J. 1:841-845, 1982; Ausubel et 
al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, 
Inc., New York, 1987; Hawley-Nelson et al., Focus 15:73-79, 1993; Hagen et 
al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; 
Ringold, U.S. Pat. No. 4,656,134; Foster et al., U.S. Pat. No. 4,959,318; 
Cregg, U.S. Pat. No. 4,882,279; Stroman et al., U.S. Pat. No. 4,879,231; 
McKnight et al., U.S. Pat. No. 4,935,349; Guarino et al., U.S. Pat. No. 
5,162,222; Bang et al., U.S. Pat. No. 4,775,624; WIPO publication WO 
94/06463; Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987; Lambowitz, 
U.S. Pat. No. 4,486,533; Sambrook et al., eds., Molecular Cloning: A 
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, 1989; Goeddel 
et al., U.S. Pat. No. 4,766,075; and Baird et al., U.S. Pat. No. 
5,155,214, which are incorporated herein by reference in their entirety. 
Suitable expression vectors and host cells are widely available from 
commercial suppliers. 
Secretion of the ob protein can be enhanced by substituting a synthesized 
secretory peptide, for example, a secretory peptide derived from that of 
human tissue-type plasminogen activator (t-PA) for the native ob signal 
peptide. t-PA secretory peptides are known in the literature, see, for 
example, Rickles et al., J. Biol. Chem. 263:1563-1560, 1988 and Feng et 
al., J. Biol. Chem. 265:2022-2027, 1990. 
The expressed recombinant protein is isolated from the host cells using 
conventional purification methods, such as affinity chromatography, ion 
exchange chromatography, reverse-phase high performance liquid 
chromatography and size exclusion chromatography. It is preferred to 
purify the protein to &gt;80% purity, more preferably to &gt;90% purity, even 
more preferably &gt;95%, and particularly preferred is a pharmaceutically 
pure state, that is greater than 99.9% pure with respect to contaminating 
macromolecules, particularly other proteins and nucleic acids, and free of 
infectious and pyrogenic agents. Preferably, a purified protein is 
substantially free of other proteins, particularly other proteins of 
animal origin. 
The present invention provides an ob protein preparation in a homogeneous 
form. The substantially purified ob protein of the present invention was 
prepared using affinity chromatography (about 85% pure) and HPLC, and is 
characterized by an M.sub.r =16,482.+-.500 daltons, as determined by mass 
spectroscopy. This purified protein exhibits migration differences on 
SDS-polyacrylamide gel electrophoresis under non-denaturing versus 
denaturing conditions, confirming the presence of a disulfide bond. The ob 
protein of the present invention is provided at least 90% pure with 
respect to other contaminating proteins, as determined by HPLC analysis. 
The purified ob protein caused appetite suppression in the mouse bioassay 
(81.+-.3% BL at a dose of 13-17 .mu.m per mouse recipient. 
Purification of ob protein has also been achieved using a combination of 
chromatography methods, including affinity chromatography, Q-Fast Flow 
Sepharose, MonoQ resin, FPLC, phenyl Sepharose, hydroxyapatite, Mono S 
and/or S-Sepharose. For some preparations, it is preferable to use 
borate-based buffers, preferably about 1-100 mM borate, and more 
preferably 10 mM borate, pH=7.4. In another preferred embodiment, it is 
preferred to prepare substantially pure preparations of ob protein in the 
presence of trace metals. Suitable trace metals in this regard include 
ZnCl.sub.2, CaCl.sub.2, Na.sub.2 MoO.sub.4, CuSO.sub.4, and FeCl.sub.3. 
One of skill in the art will recognize that other divalent and trivalent 
cations may also be suitable for this purpose. In a preferred embodiment, 
one or more of each of the trace metals is present at a concentration from 
about 1 to 100 .mu.M. It is preferred that the type and concentrations of 
metal(s) added enhance the biological activity of the ob protein 
preparation, as compared to a preparation that does not contain trace 
metals. 
It is convenient to express the appetite suppression factor as a fusion 
with an affinity "tag", such as a polypeptide for which an antibody or 
other specific binding agent is available. A preferred such affinity tag 
is a polyhistidine tail, which permits purification of the fusion protein 
on immobilized nickel (Houchuli et al., Bio/Technol. 6: 1321-1325, 1988). 
In prokaryotic expression systems, a maltose binding protein (MBP) fusion 
may be advantageously used as an affinity tag. If the protein is to be 
recovered from the cytoplasm or periplasm of the host cells, the cells are 
first disrupted, and a crude extract containing the protein is recovered 
and subjected to further purification steps. Secreted protein is recovered 
from cell-conditioned media, preferably after concentration of the 
conditioned media. Selection of particular fractionation steps and the 
sequence of those steps will be based in part on the type of host cell and 
the expression system (secretory vs. non-secretory) chosen. Such 
determinations are within the level of ordinary skill in the art. 
The expressed recombinant ASF protein is useful for production of antisera 
and purified antibodies. For instance, recombinant ASF (crude, partially 
purified or purified to homogeneity) may be advantageously injected into a 
subject mammal (preferably a mouse, rat or rabbit) using methods known in 
the art to lead to production of antibodies in the subject mammal. At 
appropriate intervals, the animals are bled, and polyclonal antiserum is 
obtained. Antibodies monospecific for ASF may be prepared by passing this 
polyclonal antiserum over an insolubilized matrix having purified ASF 
bound thereto, then eluting the bound antibodies. Alternatively, 
monoclonal antibodies that bind ASF may be prepared using standard 
hybridoma technologies. In addition, recombinant antibodies may be 
prepared using known techniques. The term "antibody" as used herein 
includes, but is not limited to, intact antibodies, proteolytic fragments 
of antibodies, Fv's, complementarity determining regions, single chain 
antibodies, epitope-binding domains of protein or non-protein origin, and 
the like. 
Anti-ASF antibodies have been advantageously used in a variety of protein 
detection and purification methods. An exemplary anti-ASF antibody is 
anti-ob antibody. Such antibodies are useful for 
radioimmuno-precipitation, immunoprecipitation, Western blotting, antibody 
affinity chromatography, antigen depletion and the like. For some studies, 
the immunogen (e.g., ob protein) preparation was either untreated or 
denatured prior to immunization. Because certain protein analytical 
techniques involve denaturing a protein preparation, such native/denatured 
"paired" production of antibodies enabled more precise analysis of 
antigen. 
Antibodies directed against ob protein can be advantageously used in an 
ELISA format to detect levels of ob protein in biological fluids. In an 
exemplary ELISA, a capture anti-ob protein antibody, preferably a 
monoclonal antibody, is coated onto a microtiter well. A biological fluid, 
preferably serum, is then added to the microtiter well, and if ob protein 
is present in the biological fluid, it is bound to the capture antibody. 
To optimize the ELISA, it may be advantageous to dilute the biological 
fluid in a protein-free solution, preferably to less than about 50% serum, 
and more preferably to less than about 10% serum. An anti-ob protein 
detecting antibody is then added. Preferably, the detecting antibody and 
the capture antibody bind to discrete epitopes of the ob protein, such 
that binding of the capture antibody does not affect subsequent binding of 
the detecting antibody. The detecting antibody may be directly or 
indirectly conjugated to a detectable signal or to a signal-generating 
moiety. Suitable signals in this regard include radioactive, colorimetric 
and fluorescent signals. Suitable signal-generating moieties that act on 
signal-generating substrates include horseradish peroxidase suitable 
substrates include OPD; 3,3',5,5'-tetramethylbenzidine; 
2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid diammonium salt!; 
alkaline phosphatase suitable substrates include p-nitrophenyl phosphate 
disodium salt!; and .beta.-galactosidase suitable substrates include 
O-nitrophenyl-beta-D-galactopyranoside!. Alternatively, the signal or the 
signal-generating moiety may be conjugated to a secondary antibody that is 
capable of binding to the detecting antibody, but not to the capture 
antibody. Further, signal amplification may be achieved through use of an 
avidin-biotin conjugation system. By comparison to a standard curve 
created using known amounts of purified ob protein, the level of ob 
protein in a biological fluid sample can be determined. 
Because the relationship between obesity in humans (where the equivalent of 
the ob/ob mouse has not been identified) and ob protein has not been 
elucidated, a method for measuring ob protein levels in biological fluids 
provides useful data. It has recently been reported that ob mRNA levels 
are increased in specific tissues of massively obese persons (Considine et 
al., J. Clin. Invest. 95:2986-88, 1995; Hamilton et al., Nature Med. 
1(9):953-56, 1995; and Lonnqvist et al., Nature Med. 1(9):950-53, 1995). 
However, the unavailability of an assay to measure circulating ob protein 
levels in biological fluids, such as serum, has left open the question of 
whether the ob mRNA in obese people translates into ob protein. Through 
use of the methods of the present invention, serum ob protein levels in 
obese and normal subjects was measured. Serum obtained from obese subjects 
contained about 6 times the level of ob protein that was measured in 
normal subjects. The methods of the present invention thus enables 
investigators and clinicians to answer the important question of whether 
obesity can be correlated to non-functional ob protein, to a defect in the 
putative receptor for the protein or to a defective intermediate molecule 
in the ob-CNS feedback mechanism/pathway. 
Since two of the three obese subjects analyzed by ELISA in Example 16 had 
been diagnosed with Type II diabetes, circulating ob protein may be 
correlated with diabetes, and/or with serum insulin or glucose levels. 
Also, elevated circulating ob protein levels may be an indicator of a 
pre-diabetic condition or of a predisposition to diabetes. Thus, the 
assays described herein may be advantageously incorporated into a kit for 
screening or monitoring of subjects with diabetes or at risk for diabetes. 
For pharmaceutical use, appetite suppression factors are formulated for 
parenteral (e.g., intravenous, subcutaneous, intramuscular, 
intraperitoneal, intranasal or transdermal) or enteral (e.g., oral or 
rectal) delivery according to conventional methods. Intravenous 
administration will be by a series of injections or by continuous infusion 
over an extended period. Administration by injection or other routes of 
discreetly spaced administration will generally be performed at intervals 
ranging from weekly to once to three times daily. Treatment will continue 
until the desired body weight is reached, after which maintenance doses 
may be administered as necessary to regulate food consumption and body 
weight within the desired range. In general, pharmaceutical formulations 
will include an appetite suppression factor in combination with a 
pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% 
dextrose in water or the like. Formulations may further include one or 
more excipients, preservatives, solubilizers, buffering agents, albumin to 
prevent protein loss on vial surfaces, lubricants, fillers, stabilizers, 
etc. Methods of formulation are well known in the art and are disclosed, 
for example, in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack 
Publishing Co., Easton Pa., 1990, which is incorporated herein by 
reference. Pharmaceutical compositions for use within the present 
invention can be in the form of sterile, non-pyrogenic liquid solutions or 
suspensions, coated capsules, suppositories, lyophilized powders, 
transdermal patches or other forms known in the art. Therapeutic doses 
will generally be in the range of 0.1 .mu.g/kg to 1 mg/kg of patient 
weight per day, preferably 1-100 .mu.g/kg per day, with the exact dose 
determined by the clinician according to accepted standards, taking into 
account the nature and severity of the condition to be treated, patient 
traits, etc. Determination of dose is within the level of ordinary skill 
in the art. Initial dose determinations can be made by extrapolation from 
animal models according to standard principles, taking into consideration 
route of administration and pharmacokinetic factors such as rate of 
absorption, distribution, biotransformation, bioavailability and rate of 
excretion. See, for example, Goodman and Gilman, eds., The Pharmacological 
Basis of Therapeutics, Fifth Edition, MacMillan Publishing Co., Inc., New 
York, 1975. 
The ob protein, a representative appetite suppression factor, is a useful 
tool for the study of appetite control. For example, the protein can be 
used to design a probe for cloning DNA encoding its cellular receptor. 
Methods for cloning cellular receptors are known in the art. In a 
preferred method, mammalian cells (e.g., BHK 570 cells; ATCC CRL 10314) 
are transfected with a cDNA library prepared from ventromedial 
hypothalamus tissue. The transfectants are plated, and labeled appetite 
suppression factor is applied to the cell layer. Binding of ob protein 
(appetite suppression factor) is indicative of receptor expression. 
In an alternative method, degenerate primers are designed from sequences of 
known members of the cytokine receptor superfamily, based on the putative 
structural homology of ligands for the receptors of this family. Using 
pattern recognition (Cohen et al., Biochem. 25:266-75, 1986) and neural 
network (Kneller et al., J. Mol. Biol. 214:171-82, 1990) software for the 
production of secondary structure, the ob gene product was predicted to 
fold into a four alpha-helix bundle. This helical structure prediction was 
used as a guide in the generation of a mutiple alignment with other 
helical cytokines (Buzan, Immunology Today 11: 350-54, 1990; and Gribskov 
et al., PNAS USA 84: 4355-58, 1987). The multiple alignment demonstrated 
that ob protein has similarity to the helical cytokine structure class. 
Additional evidence of the possible relatedness of ob protein to the 
helical cytokine structure class was provided by the two forms of ob cDNA 
(+Gln and -Gln). These two forms may be caused by slippage at a splice 
acceptor sequence, indicating the location of a exon-intron boundary 
(Zhang et al., ibid). With respect to the multiple alignment, the location 
of the exon-intron boundary of ob was determined to be coincident with an 
exon-intron boundary in interleukin-3 and GM-CSF, and is within two 
residues of exon-intron boundaries in interleukin-2 and interleukin-6. 
For cloning an ASF receptor, genomic DNA is amplified, and overlapping, 
complementary clones are extended and amplified using primers specific for 
engineered 5' and 3' tails on the template DNA. DNA molecules encoding a 
receptor for ASF are then cloned through the application of conventional 
techniques. Such molecules are then expressed in engineered host cells to 
produce recombinant ASF receptor. The receptor, in cell-bound or cell-free 
form, is used to screen compounds for ASF agonist or antagonist activity. 
Screening methods of this type are known in the art (see, for example, 
Hendry et al., J. Immunological Methods 35:285-296, 1980; and Lam et al., 
Nature 354:82-84, 1991). In general, a test sample is assayed for the 
ability to bind to the receptor and, in a cellular system, produce or 
inhibit a biological response in the target cell. Compounds identified in 
this manner may find utility in the treatment of a variety of eating 
disorders, or may be lead compounds for further development of therapeutic 
agents. Assays of the type disclosed above are, of course, useful within 
such further development. 
The cultures listed in the following chart are deposited in the American 
Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville Md. 20852, 
U.S.A. 
______________________________________ 
Accession 
Deposited Material 
Number Date of Deposit 
______________________________________ 
Antibody producing hybridoma line 
HB-12437 November 20, 
216.1.2.1.2 1997 
Antibody producing hybridoma line 
HB-12439 November 20, 
216.3.3.2.1 1997 
Antibody producing hybridoma line 
HB-12438 November 20, 
218.5.4.4 1997 
______________________________________ 
The cultures will be maintained and access will be provided according to 
the requirements of 37 CFR .sctn..sctn. 1.806 and 1.808(a). 
The invention is further illustrated by the following non-limiting 
examples. 
EXAMPLES 
Example 1 
Methods for Establishing Recipient Test Animals 
Nine 6-8 week-old male, C57BL/6J-ob/ob (non-diabetic) mice (Jackson 
Laboratories, Bar Harbor, Me.) were isolated to separate cages with one 
animal per cage. The weight of the mice ranged from 38 to 47 grams. The 
mice were presented with preweighed quantities of chow in the range of 
14-18 grams of standard rodent chow blocks (Teklad, Madison, Wis.), water 
and given a 1 ml intraperitoneal injection of PBS at 0930-1030 hours. The 
lights were turned off until 2230 hours, reversing the normal light cycle. 
Twenty-four hours later, the uneaten chow blocks were removed and weighed. 
Each animal was removed from its cage, weighed and injected with 1 ml of 
PBS, intraperitoneally. New chow blocks were weighed and placed in each 
cage, and lights were turned off. This process was repeated for five days 
until all nine mice were identified as having consumed the same amount of 
chow, within a 10% deviation, for two feeding cycles. Table 1 shows the 
net chow weight consumed by each animal to establish the baseline. Day 1 
begins feeding program. 
TABLE 1 
______________________________________ 
Day: 2 3 4 5 6 7 
mouse # grams of consumed food 
______________________________________ 
87 5.9 6.6 7.4 6.8 6.9 5.7 
84 4.5 5.3 6.2 6.7 6.5 7.2 
78 5.7 6.4 7.8 8.5 6.7 7.1 
74 5.7 6.4 7.4 6.3 6.9 7.5 
86 5.6 6.0 6.6 7.3 6.6 6.4 
83 4.9 5.0 5.7 5.8 5.2 5.4 
79 5.9 6.3 6.6 6.9 6.2 6.8 
77 5.4 6.6 5.7 5.0 6.5 6.7 
88 4.8 4.7 6.0 5.8 6.0 6.1 
______________________________________ 
By day 7, it was established that each mouse had acceptable variation in 
food consumption, compared to consumption on day 6, and the animals were 
administered a single injection of test sample or control buffer on day 7. 
Example 2 
Adipose Conditioned Medium 
A. Preparation of Adipose Conditioned Medium 
Epididymal, inguinal, dorsal and retroperitoneal adipose tissue pads were 
removed from 12-14 week old non-fasted male fa/fa (Zucker) rats (Harlan 
Sprague Dawley Co., Madison, Wis.) under methophane anesthesia, placed 
into room temperature Dulbecco's Modified Eagle's medium (DMEM) containing 
4.5 g/l glucose, 2 mM L-glutamine and 20 mM HEPES (Sigma, St. Louis, Mo.), 
and dissected immediately after collection into 10-15 mg fragments using 
sharp scissors. Microdissected tissue fragments were washed once in medium 
and incubated in 150 mm culture dishes (American Scientific Products, 
Chicago, Ill.) for 3-5 h at 37.degree. C. and 5% CO.sub.2 atmosphere. 
Approximately 2.5 ml of medium was used per gram of tissue. Following 
incubation, fat was removed by centrifugation at 2200 g, and the aqueous 
layer was filtered through a 0.2 .mu.m membrane (American Scientific 
Products). The filtrate was concentrated 20 fold using a 1 kDa cutoff 
membrane and a sterile 76 mm pressure cell (YM-1 membrane, Amicon, 
Chicago, Ill.) to a volume of 3-5 ml per rat. The final protein 
concentration was determined using a Pierce BCA assay (Pierce, Rockford, 
Ill.), according to the manufacturer's specifications. 
B. Adipose Tissue Synthesizes and Secretes ASF 
Male, non-fasted db/db adipose tissue (about 2 g) was microdissected and 
placed in medium, as described in 2.A., above, containing 10.sup.-7 M 
insulin. The adipose tissue was washed once with unlabeled medium, then 
resuspended in 4 ml of medium containing 5 .mu.l EXRE.sup.35 S.sup.35 
S.RTM. Protein Labeling Kit (NEN, Boston, Mass.; a mixture of .sup.35 
S-methionine and .sup.35 S-cysteine; also designated ".sup.35 S-EXPRESS", 
herein). The adipose tissue was incubated in this medium for 210 min, and 
every 30 min a 200 .mu.l sample was removed. To each 200 .mu.l sample was 
added 15 .mu.l fetal bovine serum (FBS) as a carrier and 200 .mu.l cold 
40% trichloroacetic acid (TCA). This mixture was held on ice for 1 h, then 
filtered through a Whatman GFA glass fiber filter. The filter was washed 
with 10 ml 20% TCA, and the filters were dried and counted. 
The TCA-insoluble material derived from the .sup.35 S-labeled conditioned 
medium exhibited a linear and progressive incorporation of radioactivity 
over the full time course of the experiment. These results indicate that 
the adipose tissue is synthesizing new protein during incubation in the 
culture medium. When the time course of this experiment was extended to 20 
h, linear incorporation of .sup.35 S into TCA insoluble material was 
observed. 
To determine the effect of cycloheximide on incorporation of .sup.35 S 
label into TCA-insoluble material, adipose tissue was treated as above, 
but duplicate preparations were prepared. To one of the duplicates, 
cycloheximide (0.2 mg/ml) was added at time 0, and the experiment was run 
for 480 min. In the duplicate preparations that included cycloheximide, a 
noticeable decrease in .sup.35 S incorporation into TCA-insoluble material 
was noted. 
C. Radioimmunoprecipitation of db/db Mouse Adipose Conditioned Medium (ACM) 
An ACM radiolabeled protein preparation was obtained as follows. 
Retroperitoneal, inguinal, epididymal and dorsal fat pads were harvested 
from a 41 g female db/db C57BL/6J (Jackson Labs) mouse (8 weeks of age). 
The fat (7.8 g) was processed as described in 2.A., above, except that the 
fat was microdissected in PBS, and washed three times with 40 ml of fresh 
PBS (each wash). The fat was then suspended in 20 ml of labeling medium 
(DMEM minus methionine and cysteine, and containing L-glutamine (0.29 
mg/ml), penicillin (0.05 mg/ml), streptomycin (0.05 mg/ml), neomycin (0.01 
mg/ml), sodium pyruvate (0.1 mg/ml), 10 mM HEPES, pH 7.0, insulin (5 
.mu.g/ml), and .sup.35 S-EXPRESS (10 .mu.Ci/ml)) in a 150 mm tissue 
culture dish. The fat was radiolabeled for 5.5 h. The medium was removed 
from under the fat, sterile filtered with a 0.2 .mu.m filter, and 
concentrated using a Centriprep-3 (Amicon) device. Radiolabeled medium (15 
ml) was concentrated to 800 .mu.l at 4.degree. C. 
D. Detection of ob Protein in ACM 
Concentrated ACM (200 .mu.l) was mixed with 5 .mu.l or with 25 .mu.l 
polyclonal anti-ob antibody (2 mg/ml; raised against denatured MBP::ob 
fusion protein, as described in Example 10.B., infra). These mixtures were 
incubated on ice for approximately 3 h. 
A PANSORBIN (Calbiochem, La Jolla, Calif.) stock solution was fully 
resuspended and two 1 ml aliquots of PANSORBIN cells were withdrawn. These 
aliquots were microfuged at .apprxeq.4,000 RPM at 4.degree. C. The pellets 
were resuspended in 50 mM Tris, pH 7.4, containing 150 mM NaCl, 5 mM EDTA 
and 0.5% NP-40. This mixture was recentrifued and resuspended in the same 
solution with NP-40 at 0.05%, instead of 0.5%. Each PANSORBIN aliquot (100 
.mu.l) so prepared was mixed with an ACM/antibody mixture (either 5 .mu.l 
or 25 .mu.l Ab), and incubated for 15 min at room temperature. After this 
incubation, the PANSORBIN/ACM/antibody mixtures were centrifuged at 
4.degree. C., 10,000 RPM for 2 min. The supernatant was removed, and the 
pellet was resuspended in 30 .mu.l of 1.times. SDS-PAGE sample loading 
buffer (63 mM Tris, pH 6.8, containing 10% (v/v) glycerol, 2% (w/v) SDS, 
5% (v/v) .beta.-mercaptoethanol and 0.0125% (w/v) bromophenol blue). The 
SDS-PAGE samples were boiled for 5 min, chilled, and loaded onto a Daiichi 
10-20% glycine PAGE multigel (Integrated Separation Systems, Natick, 
Mass.), along with MULTIMARK molecular weight markers (Novex, San Diego, 
Calif.; multicolored size markers). 
The gel was electrophoresed at constant voltage (125V) until the dye front 
was approximately 1 cm from the bottom of the gel. The gel was fixed in 
methanol/acetic acid (40%/10%) for 30 min at room temperature, then soaked 
with AMPLIFY (Amersham) for an additional 30 min. The gel was then dried 
down onto blotting paper, exposed to phosphor plates for 8 h, and analyzed 
using a PHOSPHOIMAGER (Molecular Dynamics, Sunnyvale, Calif.). 
Subsequently, the gel was also exposed to X-ray film overnight. 
A discrete band migrating at the predicted molecular weight of ob protein 
was observed. This result evidences that ob protein is present in ACM. 
E. Detection of ob mRNA 
mRNA levels of ob in normal rats that had either been fasted or fed was 
examined. Adipose tissue from 16 week-old male Sprague Dawley rats (Harlan 
Sprague Dawley Co., Madison, Wis.) was collected from the following 
groups: 
1) rats fasted for 48 hours 
2) rats fasted for 72 hours 
3) rats fasted for 48 hours and refed for 24 hours 
4) rats fed ad libitum 
The RNA was isolated according the the method of Chomczynski et al., Anal. 
Biochem. 162:156-159, 1987. Northern analysis was done using 11 .mu.g of 
RNA from each group electrophoresed on 1% agarose gels and transferred to 
nitrocellulose. The nitrocellulose filters were probed using .sup.32 
P-labeled mouse ob cDNA (SEQ ID NO:1). Results, showed that ob mRNA levels 
observed in adipose tissue from the refed (group 3) and ad libitum fed 
(group 4) donor animals were approximately twice the levels observed from 
the animals fasted for 48 hours (group 1) and 72 hours (group 2). 
Example 3 
Adipose Conditioned Medium Suppresses Food Intake in ob/ob Recipient Mice 
Male BALB/c mice received two daily intraperitoneal injections (0.5 ml) of 
concentrated fa/fa rat adipose conditioned medium (see Example 2.A., 
above) having a protein concentration of 25-35 mg/ml. Comparable male 
BALB/c mice received two daily intraperitoneal injections of one of the 
following control preparations: 
(a) bovine fraction V albumin (Sigma) in PBS at a concentration of 28.6 
mg/ml or 14.3 mg/ml; 
(b) liver conditioned medium at a concentration of 45.4 mg/ml or 22.7 
mg/ml, obtained by culturing microdissected liver tissue excised from a 
500 g male Sprague-Dawley rat under the same conditions as described for 
adipose conditioned medium (see Example 2, above); 
(c) serum obtained by orbital bleeding of male db/+mice (Jackson 
Laboratory), administered as a 0.5 ml intraperitoneal dose; 
(d) BAF3 24-11 (an IL-3-dependent cell line that expresses mouse 
thrombopoietin) conditioned medium, wherein the medium per 500 ml of F-DV 
(an equal mixture of D-MEM and Ham's F-12 media), the following were 
added: 50 .mu.l transferrin (10 mg/ml); 500 .mu.l insulin (5 mg/ml); 250 
.mu.l selenium (4 .mu.g/ml); 5 ml fetuin (1 mg/ml); 5 ml sodium pyruvate; 
5 ml L-glutamine (29 mg/ml); and 12.5 ml 25 mM HEPES! tested at 6.25 
mg/ml; or 
(e) human fibrinogen in PBS at a concentration of 28.3 mg/ml or 14.15 
mg/ml. 
As shown in FIG. 1, the fa/fa adipose conditioned medium produced a 
dose-related suppression of 24 h chow intake. The maximal exceeded any 
suppression observed in recipients that received injection of a control 
preparation. 
Example 4 
Adipose Tissue Excised from Overfed Rats 
Retroperitoneal adipose tissue was collected under chloral 
hydrate/pentobarbital anesthesia from overnight-fasted Long-Evans rats 
that had been chronically overfed to produce obesity, as described by B. 
E. Wilson et al., Am. J. Physiol. 259: R1148, 1990. Two to four grams of 
adipose tissue were microdissected and incubated for 4 h at 37.degree. C. 
in 10 ml of tissue culture Medium 199 plus 15 mM HEPES and 1 .mu.g/ml 
leupeptin. Conditioned medium was concentrated by ultrafiltration (1 kD 
cutoff), and final protein concentration was determined by the method of 
Bradford (Anal. Biochem., 72:248-54, 1976). 
An aliquot of this Long-Evans adipose conditioned medium was injected into 
the third cerebral ventricle of rats trained to consume a test meal. More 
specifically, stainless steel 22 gauge cannulas were stereotaxically 
placed into the third ventricles of six 300 g male Long-Evans rats that 
were subsequently trained to consume a 2 h test meal of powdered chow at 
the onset of darkness. Chow was also presented for 6 h at the end of the 
dark phase to assure normal 24 h caloric intake and growth. Cannula 
function was confirmed by a positive drinking response to a 100 ng bolus 
injection of angiotensin II. All injections were given immediately prior 
to the beginning of the dark phase, and chow was weighed 2 h later. 
A 15 l aliquot of concentrated Long-Evans ACM (containing 50, 60, 170, 290 
or 350 .mu.g protein) was injected intracerebroventricularly into a 
cannulated recipient rat (described above). Data are expressed as a 
percentage of baseline (% BL), which was the mean 2 h chow intake 
following a 15 .mu.l injection of unconditioned Medium 199 on the day 
preceding and the day following administration of adipose conditioned 
medium. Points represent the mean.+-.SE determined using 6 rats per group 
for each protein amount injected. The line represents the best fit of 
logarithmic function to the data set (r.sup.2 +0.734, p&lt;0.05). 
The recipients of adipose conditioned medium exhibited a significant 
feeding suppression, as compared to baseline food intake (FIG. 2). The 
centrally effective Long-Evans adipose conditioned medium protein dose of 
0.35 mg in a 300 g recipient rat (e.g., 1.17 mg/kg) was about 1000-fold 
less than the peripherally effective dose of 30 mg in a 25 g mouse (e.g., 
1200 mg/kg). This central-to-peripheral dose ratio, which is expected on 
the basis of the blood-brain barrier and dilution of an endocrine factor 
in the circulation, was consistent with a central locus of ASF action. 
Example 5 
Comparison of Feeding Suppression in Various Mouse Strains 
The effect of fa/fa adipose conditioned medium on chow intake of ob/ob 
mice, db/db mice and BALB/c mice was determined. ob/ob mice of both sexes 
were obtained from Jackson Laboratories at 6-8 weeks of age and studied at 
a body weight of 45-55 g. 
Briefly, a recipient mouse was administered two daily intraperitoneal 
injections of fa/fa adipose conditioned medium at a dose indicated in FIG. 
3, and 24 h chow intake was measured (FIG. 3). ob/ob recipients are 
represented by triangles; db/db mice by circles; and BALB/c mice by 
squares. Points represent the mean.+-.SE using 2-5 mice per group for each 
protein dose injected. Greater feeding suppression was observed in ob/ob 
mice than in db/db or BALB/c mice. 
Five db/db mice received a 0.40 mg/g intraperitoneal dose of ACM. This dose 
was matched to the 0.40 mg/g median dose administered to one of the three 
groups of ob/ob recipients. The db/db recipients displayed a 24 h food 
intake that was 89.+-.5.1% of baseline level, as compared to the three 
ob/ob mice that showed a 24 h food intake that was 58.+-.5.5% of baseline 
level (p=0.01). 
Since the injected ACM diminished food intake in ob/ob mice (which are 
believed to have a deficiency in production of satiety factor), but not in 
db/db mice (which are believed to be insensitive to satiety factor), these 
results evidence a specific appetite regulating activity. Accordingly, 
these data rule out the possibility that ACM exerted a non-specific toxic 
effect, which would have affected both strains equally. 
The duration of feeding suppression in ob/ob and BALB/c recipients was also 
compared (FIG. 4). Briefly, 2 ob/ob or 3 BALB/c recipients received 2 
intraperitoneal injections of fa/fa rat ACM (protein 
concentration=28.3.+-.1.5 mg/ml) on day 2 of the assay. On day 1 and days 
3-7 of the assay, the recipients received a daily intraperitoneal 
injection of PBS. Points represent the mean.+-.SE expressed as a percent 
of baseline 24 h chow intake computed for the 3 days prior to test sample 
injection(s). The ob/ob recipients exhibited a protracted feeding 
suppression as compared to BALB/c recipients. For these reasons, ob/ob 
recipients were used in further experiments. 
Example 6 
Taste Aversion Test 
A two bottle conditioned taste aversion test of fa/fa rat adipose 
conditioned medium was performed. Briefly, overnight fasted ob/ob test 
mice or control mice (n=5 per group) were trained over a 4 d period to 
drink flavor 1 liquid diet (vanilla Ensure.TM., Abbott Labs) for 2 h 
following a 1 ml intraperitoneal injection of phosphate-buffered saline 
(PBS; pH 7.4). On day 5, test mice were injected intraperitoneally with 1 
ml of adipose conditioned medium (protein concentration=20 mg/ml) and 
control mice were injected intraperitoneally with 1 ml PBS. Both groups 
were then offered flavor 2 diet (chocolate Ensure.TM.). On day 6, all mice 
were injected with 1 ml PBS, and both flavor 1 and flavor 2 were 
presented. As a positive control, test mice received 1 ml of 0.15M LiCl in 
water, control mice received 1 ml PBS, and both groups were offered flavor 
3 diet (eggnog Ensure.TM.) on day 7. On day 8, all mice were injected with 
1 ml PBS, and both flavor 1 and flavor 3 were presented. The results are 
shown in Table 2. 
TABLE 2 
______________________________________ 
Volume Consumed (ml .+-. SD) 
Flavor 1 Flavor 2 Flavor 3 Total 
______________________________________ 
Day 6 
Test 5.9 .+-. 1.8 
2.1 .+-. 1.8 8.0 .+-. 0.36 
Control 4.1 .+-. 2.3 
3.8 .+-. 2.5 7.9 .+-. 0.46 
p* NS NS NS 
Day 8 
Test 7.6 .+-. 0.81 0.22 .+-. 0.13 
7.8 .+-. 0.86 
Control 5.1 .+-. 2.3 2.4 .+-. 1.8 
7.5 .+-. 0.64 
p* 0.05 0.03 NS 
______________________________________ 
*Unpaired 2tail t test with significance level of 0.05 
NS: not significant 
Taste aversion was not produced in test ob/ob mice by pairing injections of 
adipose conditioned medium with the presentation of a novel flavor (see 
Table 2, Day 6 results). In contrast, these same test ob/ob mice exhibited 
a taste aversion when lithium chloride, a nauseant, was paired with a 
novel flavor (see Day 8 results). 
When this experiment was repeated, there again was no significant (p&gt;0.05) 
taste aversion in test mice by pairing injections of ACM with the 
presentation of novel flavor 2. When these same test mice were examined 
for taste aversion by pairing injections of lithium chloride to flavor 3, 
a statistical difference (p=0.001) between flavor 1 and flavor 3 
consumption was observed. 
Example 7 
Effect of Various Rodent Adipose Conditioned Media on Feeding Response in 
ob/ob Recipients 
To test the hypothesis that the ob/ob mutation results in partial or 
complete loss of ASF activity, ob/ob fat conditioned medium and other 
donor rodent fat conditioned media were examined for their effect on 
feeding after injection into ob/ob recipients. Briefly, conditioned media 
were prepared as described in Example 2, above. These media were 
concentrated to the protein concentration indicated in Table 3, below, and 
were then administered to ob/ob recipients by intraperitoneal injections 
of 1 ml aliquots. Response is expressed as percent of baseline 24 h chow 
intake in n separate assays. Analysis of variance indicated significant 
intergroup variability (F=34.174, p&lt;0.0001). 
TABLE 3 
______________________________________ 
Protein Response 
Donor strain n (mg/ml) (%)* p 
______________________________________ 
ob/ob mouse 8 21.2 91.1 .+-. 1.8 
-- 
db/db mouse 4 21.0 72.3 .+-. 3.0 
0.002 
Sprague Dawley rat 
2 16.6 73.3 .+-. 3.0 
0.002 
fa/fa rat 7 21.2 52.4 .+-. 3.0 
&lt;0.0001 
______________________________________ 
*mean .+-. SE 
Table 3 shows that ob/ob donor adipose tissue conditioned medium did not 
result in a statistically significant suppression of the feeding response 
in ob/ob recipient mice. In contrast, the other rodent donor adipose 
tissue conditioned media did contain significant appetite down-regulating 
activity. In this experiment, the db/db donor mice were atypically small 
and young, and did not yield typical amounts of adipose tissue. Therefore, 
these particular db/db mice were not true matches for the other donor 
rodents tested. This fact may explain why the db/db ACM yielded responses 
that resembled those of normal rats, rather than fa/fa rats. 
Example 8 
Determination of Weight Loss Associated with Injection of Adipose 
Conditioned Medium 
The effect of daily administration of adipose conditioned medium on body 
weight and chow intake of ob/ob recipients was determined. Briefly, four 
control mice (circles; see FIG. 5) each received a daily 1 ml 
intraperitoneal injection of PBS throughout the 21 day study period. Three 
test mice (squares) received 1 ml injections of PBS through day 12, then 
the daily injections were switched to 1 ml of fa/fa adipose conditioned 
medium (protein concentration=20 mg/ml). Three mice (triangles) received 
PBS injections throughout the study, but were fed chow weights identical 
to the amount of chow consumed by test mice on the previous day, beginning 
on day 12. All points represent the mean.+-.SE. 
A single daily injection of fa/fa ACM produced a sustained reduction in 
chow intake and progressive weight loss in ob/ob recipient mice, as 
compared to chow intake and weight loss in recipient mice that received a 
single daily injection of PBS (FIG. 5). ob/ob mice that were injected with 
PBS, but fed the same amount of chow as that consumed by ob/ob recipients 
that were injected with ACM, lost weight at an identical rate. These data 
demonstrate that reduced caloric uptake accounted for the weight loss in 
recipients that were injected with ACM yet had continuous access to chow. 
Further, these results are inconsistent with weight loss attributable to 
increased energy expenditure by the recipients as a result of injection 
with ACM. 
When the ACM recipient group was switched to injection of PBS at day 25, 
the three recipient groups demontrated comparable body weights and chow 
intakes by day 45-50 and day 28, respectively. This comparability 
continued through the 60 day time course of the study. 
Example 9 
Partial Purification of ACM 
Adipose tissue was excised from 10 fa/fa (Zucker) rats and ACM was prepared 
as in Example 2.A., above. Groups of 5, 3 and 2 rats served as donors on 
different days. The ACM generated from these three groups of rat adipose 
tissue each contained about 20 mg protein/ml after concentration. The 
discrete concentrated ACMs were stored on ice until pooling. Upon 
concentration, a 1 ml aliquot of each concentrated preparation was 
intraperitoneally injected into ob/ob recipient mice, resulting in a 
feeding response of 100%, 100% and 82% of baseline, respectively). 
Briefly, 2220 ml of total ACM (conditioned using a total of 867.4 g adipose 
tissue) were concentrated by ultrafiltration, using a stirred cell 
apparatus and a 1 kD cut-off membrane (Amicon), to a a final, pooled 
volume of 100 ml. The concentrated preparation (100 ml) was dialyzed 
against 20 mM Tris buffer, pH 8.0, then passed through a 0.2 .mu.m filter 
(Gelman; Ann Arbor, Mich.). This material was then applied to a 20 ml 
Q-Sepharose.RTM. ion exchange fast flow column (Pharmacia; Piscataway, 
N.J.; cross-linked agarose matrix that binds through quarternary amino 
groups that remain equally charged throughout the pH range 2-12) that had 
been equilibrated overnight with 20 mM Tris buffer (pH 8.0; 0.2.mu. 
filtered) at a flow rate of 0.1 ml/min. The concentrated preparation (125 
ml) was loaded onto the column at 2 ml/min, washed with 20 mM Tris buffer, 
and the effluent was collected for about 65 min. The column was then 
washed for 20 minutes and the fractions independently collected. The 
column was then eluted using a 40 minutes 0-0.5M NaCl gradient. Fractions 
were monitored at A.sub.220 ; collected (5 minutes; 10 ml); pooled (2 
fractions per pool; i.e., 1+2=pool 1 and the like) through fraction 12; 
dialyzed and concentrated using an Amicon CentriPrep.TM. apparatus and a 3 
kD cut-off membrane. Fractions containing wash effluent were concentrated 
using a stirred cell apparatus and a 1 kD cut-off membrane. Aliquots of 
the concentrated pools and column effluent were tested for appetite 
regulating activity. Column effluent means the material that flowed 
through the column upon application of the concentrated preparation. 
TABLE 4 
______________________________________ 
.Pool # 
% Baseline 
______________________________________ 
1 103 
2 70 
3 90 
4 75 
5 90 
6 97 
Effluent 
97 
______________________________________ 
A 1 ml aliquot of each pool was injected into one recipient. 
Each pool volume was about 6 ml. 
Pool 2, which contained fractions 3 and 4, exhibited appetite suppression 
activity when tested in the assay described in Examples 1 and 3, above. 
These fractions eluted at a salt concentration of about 0.1-0.25M NaCl. 
Therefore, at pH 8.0, the active molecule(s) are slightly negatively 
charged, or exhibit a negatively charged region. Pool 4 (fractions 7 and 
8; about 0.38-0.5M NaCl) demonstrated appetite suppression activity, but 
was less active than pool 2. Polyacrylamide gel electrophoresis analysis 
of pools 2 and 4 showed multiple protein bands, However, several protein 
bands in the concentrated preparation were eliminated or reduced in the 
pool 2 and 4 profiles, and several bands were enriched in the pool 2 and 4 
profiles, as compared to the concentrated starting preparation. 
Pools 2-5 above were assayed for protein content, with the following 
results: pool 2=21.4 mg/ml; pool 3=41.9 mg/ml; pool 4=31.9 mg/ml; and pool 
5=10.2 mg/ml. These original pools 2-5 (6 ml each) were further 
concentrated with a CentriPrep.TM. apparatus to a volume of 2-3 ml/pool. 
These reconcentrated pools were bioassayed as above, with the following 
results: pool 2=63% of baseline chow intake (BL); pool 3=66% BL; pool 
4=72% BL; and pool 5=75% BL. Therefore, ASF activity was present in pools 
2-5 upon further concentration of the pools. 
Subsequently, another large scale batch of ACM was prepared and partially 
purified as above. Three recipient animals per pool were tested in the 
bioassay, with the following results: pool 2=73% BL (8 mg/ml protein); 
pool 3=68% BL (23 mg/ml protein); pool 4=93% BL (14 mg/ml protein); pool 
5=95% BL (not determined). These data suggest that pool 2 material 
contained the greatest ASF activity on a mg protein basis. 
Example 10 
Prokaryotic Expression of a Representative ASF 
A. Cloning and Expression of MBP:ob Fusion Protein in E. coli 
(1) mouse ob 
A representative appetite suppression factor (ASF) was expressed in E. coli 
as a maltose binding protein (MBP) fusion using an expression and 
purification kit obtained from New England BioLabs (Beverly, Mass.), 
according to the protocols supplied with the kit. Briefly, a mouse ob cDNA 
(SEQ ID NO:1) was prepared with Eco RI and Sal I adhesive ends and was 
cloned into the pMAL-c2 vector (New England Biolabs). The vector, pMALc2, 
was linearized with restriction enzymes Eco RI and Sal I, and treated with 
calf alkaline phosphatase. The vector and cDNA segments were gel purified. 
A ligation reaction containing 100 ng of purified vector and approximately 
100 ng of cDNA was prepared and the resulting construct (MBP::ob fusion) 
was introduced into E. coli MC-1061 host cells (Clontech, Palo Alto, 
Calif.). LB plates containing 100 .mu.g/ml ampicillin were inoculated with 
transformed cells. Colonies were isolated and plasmid DNA was prepared. 
Restriction analysis of the plasmid DNA identified colonies containing the 
in-frame fusion of the ob cDNA with the maltose binding protein cDNA. The 
constructs were further verified by sequence analysis. The verified clone, 
encoding ob protein with a Gln residue at position 49 (+Gln; SEQ ID NO:2), 
was designated pCZR90. An additional construct of mouse ob cDNA containing 
a nucleotide sequence (SEQ ID NO:5) that encodes ob protein without a Gln 
residue at position 49 (-Gln; SEQ ID NO:6) was constructed as described 
and designated pCZR100. 
The MPB::ob fusion plasmids were expressed by cells cultured in rich medium 
(LB broth) containing ampicillin (250 .mu.g/ml) to a cell density of 107 
cells/ml (OD.sub.600 =0.1). After 1 h of growth at 37.degree. C., 
expression of the MBP::ob fusion protein was induced by addition of 
isopropylthiogalactoside (IPTG) to a final concentration of 1 mM. Cultures 
were then incubated for an additional 3 h. Cells were recovered, disrupted 
by freezing and sonication, and the MBP-ob gene product fusion protein was 
recovered from a crude cell extract by affinity chromatography on amylose 
resin. The fusion protein was cleaved with factor Xa to liberate the ob 
gene product. 
More specifically, protein extracts were prepared from IPTG-induced and 
control, uninduced cultures containing the pCZR90, pCZR100 and pCZR101 
constructs, then analyzed by SDS-PAGE and Western blotting (see Example 
11). Cells were harvested from 1.5 ml of culture, disrupted in 200 .mu.l 
of lysis buffer (100 mM Tris, pH 7.0, 5% (w/v) SDS, 8M urea, 10% (v/v) 
glycerol, 2 mM EDTA, 0.01% bromophenol blue, 5% (v/v) 
.beta.-mercaptoethanol) and .apprxeq.200 .mu.l glass beads (Sigma; 
unwashed, 425-600 .mu.m size) by vigorous vortexing and then heating to 
65.degree. C. Cell extracts were clarified by centrifugation and 5 .mu.l 
samples were analyzed by SDS-PAGE. Coomassie blue staining of these gels 
revealed a predominant 60 kD band in induced samples that was not present 
in uninduced samples. Western blotting using anti-MBP antiserum (New 
England BioLabs) detected the induced band, demonstrating that the desired 
MBP::ob fusion protein was present in all three constructs. 
One liter of E. coli culture harboring the murine MBP::ob construct, pCZR90 
(+Gln), was prepared in "Fantastic medium" (Difco; 47.6 g powder and 4 ml 
glycerol per L H.sub.2 O). Expression and purification were performed as 
suggested by New England BioLabs (protocol #800). SDS-PAGE analysis of the 
purified MBP::ob fusion protein indicated that .gtoreq.90% of total 
protein present was the fusion protein. Subsequently, pCZR101 and pMALc2 
(vector with no cDNA insert) were similarly expressed and purified. 
Uncleaved mouse MBP::ob fusion protein (+Gln) was injected into mice, as 
described in Examples 1 and 3. MBP protein was purified using the 
manufacturer's specfication, and was injected as a negative control. 
Approximately 65 .mu.g of MBP protein and 50 .mu.g of MBP::ob protein were 
used for the injections. The MBP-treated mice demonstrated appetite 
suppression of 91.5% of baseline, and MBP::ob-treated mice demonstrated 
appetite suppression of 63% of baseline. 
(2) human ob 
The human ob cDNA was isolated by screening a human fat cell 5' STRETCH 
PLUS cDNA library (Clontech, Palo Alto, Calif.) in lambda gt11. The 
library was plated according the manufacturer's specifications. A probe 
was generated by PCR using primers from the mouse ob sequence (SEQ ID 
NO:1). The library was screened as described and a positive clone was 
identified and designated #25. The human ob sequence (SEQ ID NO:3) was 
confirmed by sequence analysis. 
DNA encoding the human ob cDNA was excised by digestion of the lambda gt11 
clone #25 with restriction enzymes and ligated into the expression vector 
Zem229R (deposited with American Type Culture Collection, 12301 Parklawn 
Drive, Rockville, Md. as an E. coli HB101 transformant and assigned 
accession number 69447). The human cDNA in Zem229R was designated phOB-25 
and included 5' and 3' untranslated regions and the coding region of the 
human ob sequence (SEQ ID NO:3). A construct containing human ob cDNA (SEQ 
ID NO: 3) prepared as described in 10.A. and designated pCZR101. 
B. Generation and Purification of Anti-ob Antibodies 
Rabbits were immunized (R and R Rabbits, Stanwood, Wash.) by initial 
subcutaneous injection of 250 .mu.g purified MBP::ob fusion (murine or 
human ob) protein in Complete Freund's Adjuvant, followed by booster 
subcutaneous injections every three weeks thereafter with 125 .mu.g 
purified MBP::ob fusion protein in Incomplete Freund's Adjuvant. After 
administration of the second booster injection, rabbits were bled 10 days 
after each boost and the serum was collected. For murine MBP::ob fusion 
protein injections, some rabbits received "native" fusion protein 
(untreated), and others received "denatured" fusion protein (heated to 
65.degree. C. in 1% (w/v) SDS in maltose-containing elution buffer (see 
Example 13) for 30 min). 
To prepare monoclonal antibodies, mice were immunized with native 
MBP::human ob fusion protein (prepared in the same manner as the immunogen 
for rabbit polyclonal antibody production). The mice received a 50 .mu.g 
primary injection, and 25 .mu.g booster injections every two weeks, for at 
least 4 weeks. Hybridomas are prepared and clones screened and scaled up 
according to standard procedures (see, for example, Hurrell (ed.), 
Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press 
Inc., Boca Raton, Fla., 1982). 
Anti-ob antibodies were prepared by the following method. Anti-MBP 
antibodies were depleted using immobilized MBP columns. More specifically, 
two columns, each containing 20 mg of purified MBP linked to 10 ml of 
CNBr-SEPHAROSE 4B (Pharmacia, Piscataway, N.J.) were prepared using 
"native" MBP (untreated). Similarly, two additional columns were prepared 
using "denatured" MBP (heated to 65.degree. C. in 1% (w/v) SDS in 
maltose-containing elution buffer (see Example 13) for 30 min) linked to 
CNBr-SEPHAROSE 4B. Rabbit antiserum raised against the native fusion 
protein was cycled through the two native MBP columns; rabbit antiserum 
raised against the denatured fusion protein was cycled through the two 
denatured MBP columns. Western blot analysis indicated that anti-MBP 
antibodies (present in either the native fusion protein antiserum or the 
denatured fusion protein antiserum) were essentially depleted. Each 
antiserum preparation retained the ability to recognize ob protein, as 
demonstrated by Western blotting. 
Anti-ob-specific antibodies were obtained through affinity purification, 
using a 4 ml column containing 10 mg of either native MBP::ob fusion 
protein or denatured MBP::ob fusion protein linked to CNBr-SEPHAROSE 4B. 
The native protein column was used for the native protein antiserum; the 
denatured protein column was used for the denatured protein antiserum. 
Western blot analysis of these affinity purified anti-ob-specific 
antibodies showed that each antibody preparation specifically recognized 
ob protein, but not MBP. Also, all anti-ob reactivity was retained on the 
affinity column, and could not be detected in the column flow-through. 
Example 11 
Mammalian Cell Expression of a Representative ASF 
A. mouse ob 
A representative ASF was expressed in the BHK 570 (ATCC CRL 10314) cell 
line. Mouse ASF (ob) cDNA (SEQ ID NO:1) was inserted in the pDX expression 
vector (disclosed in U.S. Pat. No. 4,959,318) modified by inserting a new 
Sal I site at base pair 1395, as an EcoRI-SalI fragment. The resulting 
construct, designated pDX-mOB, was co-transfected into the host cells with 
Zem229R (deposited with American Type Culture Collection, 12301 Parklawn 
Drive, Rockville, Md. as an E. coli HB101 transformant and assigned 
accession number 69447) by the calcium phosphate method (Sambrook et al. 
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor 
Laboratory Press, Cold Spring Harbor, N.Y., 1989) using 16 .mu.g pDX-mOB:5 
.mu.g Zem229R per 80% confluent 100 mm plate of cells. Transfected cells 
were grown in selection medium (growth medium plus 500 nm methotrexate; 
see Table 5) at 37.degree. C. and 5% CO.sub.2. 
TABLE 5 
______________________________________ 
Growth Medium 
______________________________________ 
500 ml Dulbecco's Modified Eagle's Medium (DMEM) 
(Gibco, Gaithersburg, MD) 
5% fetal calf serum (Hycione, Logan, UT) 
1 mN sodium pyruvate (Irvine, Santa Ana, CA) 
0.29 mg/ml L-glutamine (JRH Biosciences, Lenexa, KS) 
1.times. PSN (5 mg/ml penicillin, 5 mg/ml 
streptomycin, 10 mg/ml neomycin) (Gibco) 
______________________________________ 
Confluent transfected cells were split into 150 mm culture dishes or 100 mm 
culture dishes in the selection medium described above and grown to 
confluency. Fresh medium was added by removing spent medium, rinsing the 
dishes in serum-free medium (see Table 6), and adding fresh serum-free 
medium to the culture dishes. The medium was conditioned for two days and 
then removed for further characterization. 
TABLE 6 
______________________________________ 
Serum-free Medium 
______________________________________ 
250 ml of Ham's F12 medium (GIBCO-BRL, 
Gaithersburg, MD) 
250 ml of Dulbecco's Modified Eagle's Medium 
(GIBCO-BRL) 
0.01 mg/ml of transferrin (JRH Biosciences) 
5 .mu.g/ml of insulin (JRH Biosciences) 
0.01 mg/ml of fetuin (Sigma, St. Louis, MO) 
2 ng/ml of selenuim (Alrich, Milwaukee, WI) 
1 mM sodium pyruvate (Irvine) 
0.29 mg/ml L-glutamine (JRH Biosciences) 
______________________________________ 
Cells were utilized for larger scale production of conditioned medium by 
removing cells from approximately five 150 mm culture dishes, resuspending 
in 1.5 liters of growth medium, seeding in cell factories (Nunc, Kamstrup, 
Denmark), and allowing the cells to grow to confluency. When cells were 
confluent, the growth medium was removed and serum-free medium was added. 
When single culture dishes were used, serum-free medium did not contain 
insulin. Serum-free medium used for cell factories contained insulin. 
The ability of conditioned medium to suppress appetite in test animals was 
assayed. Two-hundred and fifty milliters of conditioned serum-free culture 
medium was recovered from 150 mm culture dishes, filtered to removed cell 
debris, and concentrated 20-fold by ultrafiltration through an Amicon YM-1 
76 mm membrane. The concentrated medium was assayed for appetite 
suppression activity as disclosed in Examples 1 and 3, above, using 
conditioned medium from cells transfected with Zem229R alone as a control 
(test samples, n=9; control samples, n=8). Feeding was reduced to 
74.+-.8.7% (mean.+-.standard deviation) of baseline in the test animals, 
compared to 95.+-.12% of baseline in controls. 
Mouse ob-transfected cells secrete ob protein that is immunoreactive with 
anti-ob antibody, as demonstrated by three methods (1-3). The ob protein 
is also detectable by silver staining alone (4). 
B. human ob 
Human ASF (ob) cDNA (SEQ ID NO: 3) was isolated without the 5' and 3' 
untranslated regions (from nucleotide 108 to to nucleotide 549 of SEQ ID 
NO:3) and was inserted in the pZem229R expression vector utilizing Eco RI 
sites. The resulting construct, designated pCBhOB27, was transfected into 
Chinese hamster ovary DG44 cells (Urlaub et al., Cell 33: 405-412, 1983) 
using LipofectAMINE.TM. (GIBCO-BRL, Gaithersburg, Md.), according to the 
manufacturer's specification. Ten micrograms of plasmid DNA was used per 
50-70% confluent 100 mm plate of cells. Transfected cells were grown in 
selection medium of .alpha.-MEM without nucleosides (JRH Sciences, Lenexa, 
Kans.) plus 250 nm methotrexate at 37.degree. C. and 5% CO.sub.2. 
Confluent transfected cells are split into 150 mm culture dishes or 100 mm 
culture dishes in the selection medium described above and grown to 
confluency. Fresh medium is added by removing spent medium, rinsing the 
dishes in CHO-S-SFM II (GIBCO-BRL) serum-free medium, and adding fresh 
serum-free medium to the culture dishes. The medium is conditioned for two 
days and then removed for further characterization, as described below. 
C. ob Protein Analyses 
(1) Radioimmunoprecipitation 
First, .sup.35 S-labeled mouse ob protein was detected by 
radioimmunoprecipitation (RIP) of transfected cell conditioned medium. 
Briefly, cells transfected with either ob or control vector were seeded 
into 6-well culture plates and grown to confluency. The culture medium was 
aspirated, and DMEM, depleted of cysteine and methionine, and containing 
1% fetal calf serum (FCS; 1 ml) and .sup.35 S EXPRESS (100 .mu.Ci/ml), was 
added to each well. The cells were incubated with the labeled medium for 6 
h at 37.degree. C. with 5% CO.sub.2. After the incubation period, the 
medium was collected and the cells were discarded. Four hundred 
microliters of radiolabeled medium was placed in a microfuge tube and 
precleared with 50 .mu.l PANSORBIN (Calbiochem). Rabbit anti-denatured 
mouse ob antibody (2.5 .mu.l; as described in Example 10.B., supra) was 
added, and the antigen-antibody mixture was incubated for 3 h on ice. 
After incubation, 40 .mu.l of PANSORBIN was added, and the mixture was 
incubated for 15 min on ice. The mixture was removed from the ice, spun in 
a microfuge for 30 sec, and the resulting pellet was washed in Penman 
Lysis Buffer (10 mM HEPES, 50 mM NaCl, 2.5 mM MgCl.sub.2, 0.3M sucrose and 
1% Triton X-100, pH 7.4) or RIPA buffer (10 mM Tris, pH 7.4, 1% sodium 
deoxycholate, 1% Triton X-100, 0.1% SDS, 5 mM EDTA and 0.15M NaCl). The 
resultant pellet was then resuspended in 20 .mu.l of standard SDS-PAGE 
sample loading buffer. For some experiments, the resultant pellets were 
resuspended in SDS-PAGE sample loading buffer under reducing or 
non-reducing conditions (.+-..beta.-mercaptoethanol). The SDS-PAGE samples 
were electrophoresed using preformed 10-20% miniplusTC SEPRAGELS 
(Integrated Separation Systems, Natick, Mass.). 
When radiolabeled protein was precipitated with affinity purified anti-ob 
antibodies raised against denatured MBP::ob fusion protein and resultant 
SDS-PAGE samples were prepared under reducing conditions, no bands were 
detected in the control vector sample, but a prominent band was seen 
(.apprxeq.14-16 kD) in the ob transfected sample. 
When radiolabeled protein was precipitated with affinity purified anti-ob 
antibodies raised against native MBP::ob fusion protein and SDS-PAGE 
samples were prepared under non-reducing conditions, a prominent band of 
.apprxeq.14 kD was detected in the ob-transfected sample that was absent 
in the control vector sample. When radiolabeled protein was precipitated 
with affinity purified anti-ob antibodies raised against denatured MBP::ob 
fusion protein and SDS-PAGE samples were prepared under non-reducing 
conditions, no prominent bands were detected in the control vector sample, 
but prominent bands were seen at .apprxeq.14-16 kD and at .apprxeq.30 kD 
in the ob transfected sample. These bands may represent monomer and dimer 
forms of ob protein expressed in BHK cells. 
(2) Western Blotting 
Second, mouse ob protein was detected by Western blotting of concentrated 
or unconcentrated ob transfected cell conditioned medium. Briefly, ob 
transfected or control vector cells were grown, as described in (1) above. 
Aliquots of conditioned medium (untreated or concentrated by 
ultrafiltration) were prepared in reducing or non-reducing SDS-PAGE sample 
preparation conditions, and electrophoresed as in (1) above. The gels were 
electroblotted onto nitrocellulose at 40V overnight (transfer buffer=56 g 
glycine and 12 g Tris base in 3.2 L H.sub.2 O and 0.8 L methanol). The 
nitrocellulose was blocked for 1 h or overnight in Western A (blocking) 
buffer (50 mM Tris, pH 7.4, 5 mM EDTA, 0.05% NP-40, 150 mM NaCl and 0.25% 
(w/v) gelatin). Rabbit polyclonal antiserum raised against denatured 
MBP::ob fusion protein (i.e., primary antibody) was added at a 1:5,000 
dilution in Western A buffer and incubated for 1 h. The dilute antiserum 
was removed, the nitrocellulose washed in Western A buffer, and goat 
anti-rabbit immunoglobulin (Ig) conjugated to horseradish peroxidase 
(i.e., secondary antibody) was added at a 1:10,000 dilution in Western A 
buffer. After 1 h, the dilute goat anti-rabbit Ig was removed, the 
nitrocellulose washed with Western A buffer, and ob protein bands were 
visualized using chemiluminescence (ECL luminescence kit; Amersham, 
according to the manufacturer's instructions) and exposure to X-ray film. 
When SDS-PAGE samples were prepared under reducing conditions and blotted 
using antiserum raised against denatured MBP::ob fusion protein, no bands 
were detected in the control vector sample, but a prominent band was seen 
(.apprxeq.14-16 kD) in the ob transfected sample. 
When SDS-PAGE samples were prepared under non-reducing conditions and 
blotted using antiserum raised against denatured MBP::ob fusion protein, 
no prominent bands were detected in the control vector sample, but 
prominent bands were seen at .apprxeq.14-16 kD and at .apprxeq.30 kD in 
the ob transfected sample. These results are consistent with those 
obtained using RIP. 
(3) Cold Immunoprecipitation (IP) 
Third, unlabeled cells were prepared and processed by the same method as 
described for .sup.35 S-RIP samples in (1) above, except that 200 .mu.l of 
unlabeled supernatant (concentrated .apprxeq.15.times.) was combined with 
rabbit antiserum raised against denatured MBP::ob fusion protein. These 
"cold IP" samples were subjected to SDS-PAGE, and then were Western 
blotted, as described in (2) above, or silver stained. 
An SDS-PAGE sample of cold IP material that was Western blotted with 
anti-denatured MBP::ob fusion protein antibodies demonstrated ob protein 
at .apprxeq.14-16 kD in the ob transfected sample, which was absent in the 
control vector sample. 
A sample of cold IP material that was precipitated with rabbit antiserum 
raised against denatured MBP::ob fusion protein; prepared using SDS-PAGE 
sample loading buffer containing .beta.-mercaptoethanol (reduced); then 
electrophoresed and silver stained, exhibited a prominent band at 
.apprxeq.14-16 kD in the ob transfected sample, which was absent in the 
control vector sample. Similar ob and control samples that were 
precipitated with rabbit antiserum raised against denatured MBP::ob fusion 
protein; prepared using SDS-PAGE sample loading buffer without 
.beta.-mercaptoethanol (unreduced); then electrophoresed and silver 
stained, showed a prominent band at .apprxeq.30 kD in the ob transfected 
sample, which was absent in the control vector sample. 
(4) Silver Staining 
When concentrated, ob-transfected conditioned medium and concentrated, 
control vector conditioned medium were prepared using SDS-PAGE sample 
loading buffer containing .beta.-mercaptoethanol (reduced), then 
electrophoresed and silver stained, a prominent band was visible at 
.apprxeq.14-16 kD in the ob transfected sample, which was absent in the 
control vector sample. 
D. Analyses of Additional Metabolic Effects 
The effect of chronic intraperitoneal administration of recombinant mouse 
ob protein on recipient serum insulin and glucose levels was determined. 
Briefly, orbital blood was collected from ob/ob mice before and 
immediately following 2 weeks of daily 80 .mu.g injections of recombinant 
mouse ob protein (n=6) or of ZEM control medium (n=3). Insulin levels in 
the mice that received recombinant ob protein decreased from 122.+-.11 
ng/ml at day 0 to 10.+-.1.9 ng/ml at day 14 (p=0.0002); glucose levels 
decreased from 489.+-.44 mg/dl to 167.+-.8.9 mg/dl (p=0.0007). Insulin 
levels in the mice that received the control ZEM medium preparation 
decreased from 427.+-.114 ng/ml at day 0 to 275.+-.68 ng/ml at day 14 (not 
statistically different); glucose levels increased from 427.+-.81 mg/dl to 
448.+-.87 mg/dl (not statistically different). Thus, administration of 
recombinant ob protein provides a salutory effect on insulin sensitivity 
in recipient mice. These data may reflect a direct effect of exogenous ob 
protein on insulin sensitivity, or may represent an indirect effect of 
injected ob protein activity (since ob protein induced weight loss in 
recipients that were injected with ob protein). 
Example 12 
Mammalian Expression of His-Tagged ASF 
A His-tagged mouse ob cDNA construct was expressed in BHK 570 cells. An 
inframe tract of six histidine codons was added to the 3' end of the ob 
coding sequence by PCR using the mouse PCR-generated cDNA as template. The 
resulting PCR product was inserted into the vector pHZ-200, a vector 
comprising the mouse metallothionein-1 promoter, the bacteriophage T7 
promoter flanked by multiple cloning banks containing unique restriction 
sites for insertion of coding sequences, the human growth hormone 
terminator and the bacteriophage T7 terminator. In addition, pHZ-200 
contains an E. coli origin of replication, a bacterial beta-lactamase 
gene, a mammalian selectable marker expression unit comprising the SV40 
promoter and origin, a dihydrofolate reductase gene and the SV40 
transcription terminator. The resulting plasmid was transfected into BHK 
570 cells. Transfectants were selected in 1 lM methotrexate. The tagged ob 
protein was isolated from conditioned culture medium by passing the medium 
over a column of metal chelation resin containing immobilized Ni2+ 
(HIS-BIND.TM., Novagen, Madison, Wis.). Bound protein was eluted from the 
column with 500 mM imidazole. 
An inframe tract of six histidine codons and a 4 amino acid spacer (Gly Gly 
Ser Gly) were added to the 5' end of the mature ob coding sequence by PCR 
using the mouse cDNA described above. The resulting PCR product was 
inserted into the vector pHZ-200 along with a 100 base pair fragment 
containing the tPA signal sequence for efficient secretion of the ob 
protein. The resulting plasmid was transfected into BHK 570 cells, and the 
5' tagged protein was isolated as described above. Western blot analysis 
of the N-terminal His-tagged protein showed that the major product was a 
doublet of bands at approximately 18 kD, with the C-terminal His-tagged 
protein appeared as a doublet at about 16 kD. 
The N-terminal and C-terminal tagged ob proteins were assayed for activity 
in mice (as described in Examples 1 and 3). C-terminal tagged ob protein 
did not demonstrate significant appetite suppression, while N-terminal 
protein suppressed appetite to 70% of baseline, suggesting that 
modification of the C-terminus inactivated the ob protein. The effect of 
the single injection of 25 .mu.g of N-terminal tagged protein, as shown in 
FIG. 6, was prolonged suppression of appetite when compared to unmodified 
ob produced by BHK cells. 
Example 13 
Purification of ob Protein 
A. Purification From MBP::ob Fusion Protein Expressed in Prokaryotic Cells 
(1) Amylose Column Chromatography of MBP::ob Protein 
MBP::ob fusion protein was induced in transformed E. coli with IPTG, as 
described in Example 10 above, and purified by amylose column affinity 
chromatography. 
Briefly, induced cells were collected and the weight of the cell pellet was 
determined. The cell pellet was resuspended in lysis buffer (column buffer 
(20 mM Tris, pH 7.4 containing 200 mM NaCl) containing 5 mM EDTA, 10 mM 
.beta.-mercaptoethanol and 2 mM PMSF), adding 10 ml lysis buffer per gram 
of cell pellet. The cell suspension was placed on ice and sonicated for a 
1 min period in five separate bursts, with a 5 min wait between each 
sonication burst. If the volume of the cell suspension was greater than 50 
ml, the sonication was done in six to seven 1 min bursts, or in five 2 min 
bursts. The sonicated preparation was then centrifuged at 15,000 RPM for 
30 min at 4.degree. C. The supernatant was decanted, and the pellet 
discarded. 
A 50 ml amylose resin column (2.5 cm.times.9.4 cm) was equilibrated in 
column buffer (20 mM Tris, pH 7.4 containing 200 mM NaCl) at 4.degree. C. 
This volume of resin was calculated to be sufficient to bind at least 150 
mg of fusion protein. The post-sonication supernatant was loaded onto the 
column at a rate of 1 ml/min. The loaded column was washed with 10 column 
volumes of column buffer, and then connected to a fraction collector. The 
column was eluted with elution buffer (column buffer containing 10 mM 
maltose) at 1 ml/min, and fractions were collected every 5 min. Typically, 
a peak started at about fraction 5 and continued for about 8-10 fractions. 
Elution was continued until the absorbance at 280 nm was baseline. 
The peak fractions (&gt;0.7 OD) were determined by monitoring at 280 nm and at 
320 nm. The A.sub.320 reading was subtracted from the A.sub.280 reading, 
to compensate for light scattering. The peak fractions were pooled, and 
protein concentration was determined by A.sub.280 -A.sub.320 absorption. 
MBP, E=1.48 Au(cm.sup.-1)((mg/ml).sup.-1); MBP::ob, E=1.16 Au(cm.sup.-1) 
((mg/ml).sup.-1) 
MOB=0.157 calculated and 0.489 by amino acid analysis 
The fusion protein was estimated to be about 85% pure after amylose column 
purification, as determined by SDS-PAGE densitometry. 
The protein was concentrated to about 6 mg/ml using Amicon 10K CentriPreps, 
or applied to a hydroxyapatite column. The amylose column was regenerated 
by removing the column from the cold room and washing: (i) with 2-3 column 
volumes of deionized (DI) water; (ii) with 3 column volumes of 0.1% SDS; 
(iii) with 10 column volumes of DI water; and (iv) with 2 volumes of 
column buffer. 
(2) Factor Xa Cleavage of the MBP::ob Fusion Protein 
The MBP::ob fusion protein contains a Factor Xa proteolytic cleavage site 
near the site of fusion. Upon Factor Xa cleavage, the ob protein, with a 4 
amino acid (Ile-Ser-Glu-Phe; ISEF) leader, was released. 
Briefly, the fusion protein can be cleaved in elution buffer. However, if 
cleaved MBP was to be removed from the cleaved mixture by re-passage over 
an amylose column, free maltose (present in the elution buffer) was 
removed from the purified MBP::ob preparation prior to cleavage. 
Bovine Factor Xa (1% (w/w)) was combined with purified MBP::ob fusion 
protein in 20 mM Tris, pH 7.0, containing 200 mM NaCl. This cleavage 
solution was incubated at 20.degree. C. for 18 h with gentle agitation or 
rocking. The solution was then centrifuged for 15 min at 10,000 RPM at 
4.degree. C. to remove denatured protein. 
The decanted supernatant was a mixture of: (i) correctly folded ob protein; 
(ii) incorrectly folded ob protein; (iii) multimeric species of ob 
protein; (iv) released MBP; and (v) about 9% uncleaved MBP::ob protein. 
About 1/3 of the ob protein was soluble; the remainder formed a 
precipitate. Most of the MBP was found in the soluble fraction, whereas 
the uncleaved MBP::ob protein was mainly found in the precipitate. 
(3) Purification of ob Protein 
The cleaved ob protein was purified by reverse phase chromatography using 
either a C4 or C8 column. Other reverse phase chromatography matrices may 
be suitable. 
Specifically, the Factor Xa-cleaved ob protein was added to 10 mM Tris, pH 
7.0, containing 5M freshly made urea and 100 mM .beta.-mercaptoethanol, 
and incubated at 50.degree. C. for 30 min. The reduced, unfolded protein 
was loaded onto a Vydac #214TP1022 C4 (2.2 cm.times.25 cm, 5.mu., 300 
.ANG.) column at 10 ml/min. The column was previously equilibrated in 27% 
acetonitrile (Acn)/0.1% trifluoroacetic acid (TFA). A 60 min gradient was 
started from 27% Acn/1% TFA to 72% Acn/1% TFA at a flow rate of 10 ml/min. 
The chromatography was monitored at 215 nm, 2 OD full scale, and 0.5 min 
fractions were collected. The column volume was about 80 ml. MBP protein 
eluted at 37.8% Acn, followed by uncleaved MBP::ob fusion protein at 46.6% 
Acn and ob protein at 47.6% Acn. Amino acid sequencing data, amino acid 
analysis, and mass spectroscopy data all confirmed the predicted 
composition as ISEF-ob protein. 
The precipitate and the soluble material that result after Factor Xa 
cleavage can be purified as a combined preparation or as separate (soluble 
and insoluble) fractions. Purification of ob protein from the precipitate 
yielded ob protein of higher purity, probably because the large MBP peak 
in the soluble fraction tailed into the desired ob peak. 
Human ob protein and mouse ob protein (native, non-fusion form) expressed 
in BHK cells were purified using the same method. These proteins exhibited 
slightly different column retention times that were consistent with the 
absence of the ISEF leader sequence in these ob protein preparations. 
(4) Refolding and Oxidation of ob Protein Thiols 
Pooled ob protein, present in C4 reverse phase chromatography fractions 
(prepared in A.(3), above) was lyophilized to remove Acn/TFA. The 
lyophilized preparation was redissolved in argon-sparged 10 mM Tris, pH 
7.0, containing 5M urea, and the ob preparation was then reduced using a 
20.times. excess (M/M thiols) of EKATHIOL resin (Ekagen, Menlo Park, 
Calif.; resin that contains a thiol red/ox functional group that reduces 
peptide thiol groups). Standard reducing agents, such as dithiothreitol, 
.beta.-mercaptoethanol, and glutathione, may be substituted for EKATHIOL 
resin. 
After 1 h at room temperature, the resin was removed and a 20.times. excess 
of EKATHIOX resin (Ekagen; resin contains a thiol red/ox functional group 
that oxidizes peptide thiol groups) was added. The resin was removed and 
the supernatant was dialyzed against 200 volumes of argon-sparged 10 mM 
Tris, pH 7.0, for 18 h. About 50% of the ob protein was soluble after 
dialysis, with about 9-13% present in dimer or multimer form; the 
remainder was present in monomer form. SDS-PAGE showed that ob protein 
migrated faster in non-reduced form than in reduced form, indicating that 
it is intra-disulfide bonded. The resultant purified protein was active in 
the ob bioassay (recipients described in Example 1 received one 
intraperitoneal injection of 55 .mu.g purified ob protein; mean % of 
baseline=71.+-.22; n=3). 
The insoluble precipitate that results after Factor Xa cleavage of MBP::ob 
fusion protein was resubjected to this reduction/oxidation process, and 
provided about the same % yield of refolded protein. 
(5) Separation of Monomer and Dimer/Multimer Forms 
Monomeric ob protein was separated from dimer/multimer forms of ob protein 
by chromatography on a C4 column, as described in A.(4), above. The ob 
dimer eluted at 47.6% Acn; the ob monomoer eluted at 52.8% Acn. This 
repurified, monomeric protein, which contained about 2% ob dimer, was 
freely soluble in 10 mM Tris, pH 7.0, after lyophilization. This 
repurified, monomeric ob preparation displayed equivalent activity in the 
ob bioassay as the original, refolded material. 
B. Purification of ob Protein Expressed in Mammalian Cells 
(1) Q-Fast Flow Sepharose Chromatography 
BHK cells expressing mouse ob protein (BHK/ob) produced conditioned medium 
that was sterile filtered and 10.times. concentrated. This concentrated 
material was buffer exchanged into 20 mM Tris, pH 8.0 buffer, and the 
resultant material had a conductivity of 1 milliSeimen. Optionally, the pH 
of this material was raised to pH 8.5 to improve binding to Q-Fast Flow 
Sepharose (Pharmacia). At this pH, the material transported through the 
column as a tight band during loading. The rate of transport through the 
column determined the sample volume that was loaded. 
Upon completion of sample loading, elution was aided by initiating a 
gradient between (i) 20 mM Tris, pH 8.5 and (ii) 20 mM Tris, pH 8.5, 
containing 1M NaCl. Typically, for a 100 ml Q-Fast Flow Sepharose column, 
a flow rate of 10 ml/min was used, with the gradient developed from 0% to 
30% 1M NaCl over 30 min. The ob protein eluted (with contamination) as a 
large, almost "square wave", peak. A slight degree of separation between 
monomer and dimer ob protein was observed, with the dimeric form eluting 
as slightly larger than the monomer form. 
(2) MonoQ Chromatography: Monomer and Dimer Separation 
Monomer and dimer forms of ob protein were resolved using FPLC (fast 
protein liquid chromatography) and MonoQ resins. The same buffer 
conditions as described for Q-Fast Flow Sepharose (B.(1), above) were 
employed, but lower flow rates and less steep gradients were beneficial. 
Typically, a flow rate of 1 ml/min and a gradient from 0% to 20% of 1M 
NaCl over 30 min separated the monomer and dimer species of ob protein. 
(3) Phenyl Sepharose Chromatography 
Alternatively, concentrated BHK/ob conditioned medium was chromatographed 
using phenyl Sepharose (Pharmacia), with an acceptable resultant 
purification. Briefly, concentrated BHK/ob conditioned medium was adjusted 
to pH 5.7 in 20 mM MES (morpholinoethanesulfonic acid) containing 1M NaCl. 
This material was chromatographed using a column of phenyl Sepharose that 
had been previously equilibrated in the same buffer (20 mM MES, pH 5.7, 
containing 1M NaCl). Some major contaminants "passed through" during 
sample loading, but ob protein was bound to the matrix. The column was 
then washed with starting buffer, and some additional contaminants were 
eluted, while ob protein remained bound. The bound ob protein was eluted 
with 20 mM Tris, pH 8.0. The material eluted was significantly purified 
over the starting material, particularly by removal of a very prominent 
contaminant. 
(4) Hydroxyapatite Chromatography 
In yet another alternative, concentrated BHK/ob conditioned medium was 
adjusted to pH 6.8 in 5 mM MES buffer. This material was passed over a 
column of hydroxyapatite that had been previously equilibrated in the same 
5 mM MES, pH 6.8 buffer. Most of the proteins, including all of the ob 
protein, were bound to this matrix during sample loading. The column was 
then washed with starting buffer, prior to elution of ob protein. The ob 
protein eluted at a "step" to 10 mM potassium phosphate (KP) buffer, pH 
6.8. Other major contaminants eluted at 50 mM KP to 300 mM KP. The ob 
protein obtained by this method was considerably purified compared to the 
starting material. 
C. Direct Capture and Purification of ob Protein from Unconcentrated BHK 
Conditioned Medium 
Recombinant mouse ob protein was directly captured from 1.times. BHK/ob 
cell factory medium. Briefly, the BHK/ob conditioned medium was adjusted 
to 1M in NaCl, 20 mM in MES, and pH 5.7 (with 2N NaOH). The protease 
inhibitor phenylmethylsulfonyl-fluoride (PMSF) was added to a final 
concentration 0.6 mM. The adjusted medium was pumped through a 0.45 micron 
filter onto a bed of Fast Flow Phenyl Sepharose equilibrated in 1M NaCl, 
20 mM MES, pH=5.7 (equilibration buffer). A 500 ml column with an 11.0 
inch diameter was loaded at 60 ml/min. The ob protein sticks tightly to 
the column under these conditions. Upon completion of sample loading (up 
to 30 liters of 1.times. medium), the column was washed with 5 column 
volumes of equilibration buffer. Bound proteins were then eluted with 5 mM 
borate buffer at pH=8.5. During elution, the flow rate was reduced to 12 
ml/min, and 18 ml fractions were collected. The ob protein eluted as a 
symmetric peak between fractions 40 and 60. The recovery of ob protein was 
greater than 95%, judged by analytical HPLC on a C4 reverse phase column. 
The pooled material from the Phenyl Sepharose step had a conductivity of 
about 8 milliSeimens. This pooled material was adjusted to 20 mM MES, and 
the pH was titrated to 6.8. A 2.times.5 cm column packed with 80 micron 
ceramic hydroxyapatite was equilibrated in 0.1M NaCl, 20 mM MES, pH=6.8 
buffer. The protein was passed through the column bed at 2 ml/min, and the 
effluent was collected. When the sample load was completed, the column was 
washed with equilibration buffer, with continued effluent collection, 
until the absorbance returned to baseline. Reverse phase (C4) HPLC 
demonstrated high recovery and substantial purification of ob protein. At 
this point, however, the material was only partially purified. 
If ob protein retains biological activity at acid pH (.apprxeq.pH 5.0), 
MonoS.RTM. (charged sulfonic groups that remain negative over a pH range 
of 2-12, attached to a pH-stable, beaded hydrophilic resin; Pharmacia) ion 
exchange is used for purification of ob protein. S-Sepharose.RTM. 
(sulfopropyl Sepharose; Pharmacia) tightly bound ob protein at pH=4.9, 10 
mM acetate buffer. The bound proteins were eluted with an ionic strength 
gradient, and ob protein eluted at fairly high ionic strength (i.e., at 
300-500 mosm in NaCl). These data suggest that ob protein is amenable to 
MonoS purification. Alternatively, following determination of low pH 
protein activity/stability, the bound proteins are eluted using an 
ascending pH gradient The ob protein is expected to start eluting at about 
pH=7.0. 
D. Effect of Metal Ions 
Some data suggest that metal ions complex with the ob protein. Briefly, ob 
protein from the Phenyl Sepharose pool material was dialyzed into 
borate-buffered saline (120 mM NaCl, 2.7 mM KCl, and 10 mM boric acid, 
pH=7.4), with and without "trace metals" added. The borate buffer with 
trace metals added contained the following: 10 .mu.M ZnCl.sub.2 ; 17 .mu.M 
CaCl.sub.2.2 H.sub.2 O; 8 .mu.M Na.sub.2 MoO.sub.4.2 H.sub.2 O; 5 .mu.M 
CuSO.sub.4 ; and 100 .mu.M FeCl.sub.3.6 H.sub.2 O. Under these conditions, 
some of the FeCl.sub.3 trace metal component precipitated out of solution. 
The biological activity of the ob protein pool material that was dialyzed 
against borate-buffered saline plus trace metals was significantly higher 
than that of the same material dialyzed against non-metal-containing 
borate buffer. When borate-buffered saline plus trace metals (vehicle) was 
tested in the mouse bioassay, no suppression of chow intake was observed. 
The particular metal(s) and concentrations that enhance recombinant ob 
protein acitivity are determined using routine procedures known in the 
art. 
Example 14 
Yeast Expression of a Representative ASF 
A. Cytoplasmic ob Construct 
The plasmid, designated pCZR108, was constructed to express a cytoplasmic 
form of the human ob protein. It consists of the ADH4.sup.c promoter, the 
coding region for the mature portion of the human ob protein (amino acid 
residue 22 to amino acid residue 167 of SEQ ID NO: 3), and the TPI 
terminator. The pUC-based plasmid pMVR1 (FIG. 7) was linearized with Sna 
BI and Sal I restriction enyzmes. The yeast ADH4.sup.c promoter (Ciriacy 
et al., Molec. Gen. Genet. 176:427-431, 1979) was isolated as a 1250 base 
pair Bam HI (blunted with T4-polymerase) to Eco RI fragment. A 450 base 
pair coding region of the mature form of the human ob protein (from 
nucleotide 108 to to nucleotide 549 of SEQ ID NO:3) was isolated as an Eco 
RI to Sal I fragment. The 90 base pair TPI terminator present in pMvR1 as 
a Sal I and Bgl II fragment. The pMVR1 vector was digested with Sna BI and 
Eco RI. The linearized vector was ligated with the ADH4.sup.c promoter 
fragment that had been blunted using T4 polymerase. The resulting plasmid 
was designated pCZR105 and contained the TPI terminator from pMVR1. The 
mature human ob coding region was amplified using PCR with primers ZC8769 
(SEQ ID NO: 7) and ZC8699 (SEQ ID NO: 8) to introduce Eco RI and Sal I 
sites. The amplified DNA was gel purified and ligated into pCZR105 that 
had been linearized by digest with Eco RI and Sal I. The resulting 
cytoplasmic human ob protein expression cassette plasmid was designated 
pCZR107. 
pCZR107 was subcloned as a Bgl II fragment (from pCZR107) into the Bam HI 
site of the POT vector pCZR12 to give pCZR108. pCZR12 is derived from 
pDPOT (deposited with the American Type Culture Collection as ATCC No. 
68001); it carries a wild-type LEU2 gene instead of the LEU2-D allele in 
pDPOT. 
B. Secreted ob Construct 
Plasmids pCZR112 and pCZR113 were constructed to express secreted forms of 
the human ob protein. The plasmids were made as fusions to the yeast 
.alpha.-Factor prepro segment. In pCZR112, the region encoding the Lys-Arg 
residues at the end of the .alpha.-Factor prepro segment are joined 
directly to the region encoding the mature N-terminal residues of the 
human ob protein. In pCZR113, codons that encode a Glu-Ala dipeptide are 
inserted between the .alpha.-Factor prepro Lys-Arg and the N-terminal 
residues of the human ob protein. 
The expression cassettes in plasmids pCZR112 and pCZR113 each contain: 1) a 
900 base pair TPI promoter fragment from pMVR1; b) a 250 base pair 
.alpha.-Factor prepro region; c) a 450 base pair human ob protein coding 
region and 4) a 90 base pair TPI terminator fragment from pMVR1. 
Val-Pro-Ile-Gln-Lys begins at amino acid residue 22 of SEQ ID NO: 4 and is 
the amino acid sequence of the mature N-terminus of the human ob protein. 
Using a polymerase chain reaction and the oligonucleotide primers ZC8702 
(SEQ ID NO: 9) and ZC8699 (SEQ ID NO: 8) a plasmid with the 5' sequence of 
Lys-Arg was generated and designated pCZR110. A second plasmid, designated 
pCZR111, was generated by polymerase chain reaction using the 
oligonucleotides ZC8701 (SEQ ID NO: 10) and ZC8699 (SEQ ID NO: 8), and has 
a 5' sequence of Lys-Arg-Glu-Ala. The oligonucleotide ZC8699 (SEQ ID NO: 
8) introduces a Sal I site at the 3' end of the human ob sequence. 
The two secreted forms of the human ob expression vectors were made by 
first subcloning the 250 bp Eco RI-Hind III alpha factor prepro segment 
and the 100 bp Hind III--Sal I coding region from the 3' end of the human 
ob cDNA into Eco RI and Sal I digested pMVR1.sup.H- in a three part 
ligation (pMVR1.sup.H- is a modified version of pMVR1 lacking Hind III 
sites). This intermediate plasmid was called pCZR104. The two secreted 
form of the human ob coding sequence were amplified by PCR with primers 
ZC8702 (SEQ ID NO: 9) and ZC8699 (SEQ ID NO: 8) or ZC8701 (SEQ ID NO: 10) 
and ZC8699 (SEQ ID NO: 8). pCZR104, was linearized with Hind III, and PCR 
fragments encoding the secreted forms of human ob that had been digested 
with Hind III were inserted into this site to generate the full length 
secreted human ob protein expression cassettes. 
The expression cassettes from plasmids pCZR110 and pCZR111 were subcloned 
as Bgl II fragments into the Bam HI site of pCZR12 to give pCZR112 and 
pCZR113, respectively. 
C. Expression of Yeast ob 
Plasmids pCZR108, pCZR112, and pCZR113 were transformed into Saccharomyces 
cerevisiae strain SF838-9D.sub.-- tpi (ade6, his3, leu2, ura3, tpi). 
Glucose.sup.+ Leu.sup.+ transformants were analyzed for ob expression by a 
colony blot procedure. Transformants were picked and used to inoculate 
YEPD plates, overlayed with nitrocellulose filters, and grown overnight at 
30.degree. C. Filters were exposed to 0.2N NaOH, 0.1% SDS, 35 mM 
dithiothreitol for 30 min to lyse cells. All filters were blocked with 5% 
non-fat milk (NFM) in TTBS (0.1% Tween 20, 20 mM Tris pH 7.5, 160 mM NaCl) 
for 30 min, probed with a 1/1000 dilution of affinity purified rabbit 
anti-murine ob (as described in Example 10) for one hour, and immune 
complexes were detected with a 1/1000 dilution of commercially available 
HRP-goat anti-rabbit (Bio-Rad, Richmond, Calif.). Filters were exposed by 
enhanced chemiluminescence (ECL; Amersham, Arlington Heights, Ill.), 
according to manufacturer's specifications. Human ob positive colonies 
were selected for further characterization. Yeast strains transformed with 
pCZR108 were designated CZY108, strains transformed with pCZR112 were 
designated CZY112, and strains transformed with pCZR113 were designated 
CZY113. 
YEPD shake flask cultures were grown of CZY108, CZY112, and CZY113. 1% 
ethanol was added to the CZY108 culture 2 hours prior to harvest, and 1% 
glucose was added to the CZY112 and CZY113 cultures 4 hours prior to 
harvest. The yield of cells from these cultures was 20 g/L wet cell 
weight, which corresponds to 2 g/L total cell protein. Culture medium was 
analyzed directly by SDS-PAG after being mixed 1:1 in protein sample 
buffer (5% SDS, 8M urea, 100 mM Tris pH 6.8, 2 mM EDTA, 10% glycerol) or 
treated with Endoglycosidase H (NEB) by adjusting the media to 0.5% SDS, 
50 mM sodium citrate pH 5.2 according to the manufacturer's suggestions. 5 
.mu.l aliquots of media, corresponding to 100 .mu.g of cells and 10 .mu.g 
of total cell protein, were analyzed in all cases. Intracellular fractions 
were generated by glass bead disruption of 0.25 g cell pellet in 2.5 mls 
lysis buffer (20 mM Tris pH8.0, 400 mM NaCl, 1 mM PMSF, 2 mM EDTA, 0.1 
.mu.g/ml each of leupeptin, pepstatin, and aprotinin). Protein 
concentrations in lysates were determined by Lowry assay, and 10 .mu.g 
were analyzed by SDS-PAGE, providing direct comparison between 
intracellular and extracellular levels of ob protein. For crude 
subcellular fractionation, Triton X100 was added to intracellular lysates 
to a final concentration of 2%, lysate was spun at 20,000.times.g for 20 
min, and equal volumes of clarified lysate and pellet fraction resuspended 
in protein sample buffer were analyzed. Commercially available 10-20% 
SDS-PAGE gels (Daiichi, Tokyo, Japan) were used to separate and analyze 
protein samples. The gels were stained directly with Coomasie brilliant 
blue or Western blotted. Human ob protein on Western blots was detected 
with affinity purified rabbit anti-murine ob Ab in NFM-TTBS as described 
for colony immunoblotting above. 
Transformant CZY108 produced high expression levels of human ob protein; 
whereas the untransformed control strain did not exhibit cross-reacting 
protein material. Comparison between Coomasie stained total protein of 
control cultures and CZY108 strains showed that the CZY108 strains produce 
an abundant .sup..about. 15 kD protein. Western analysis demonstrates that 
this protein was human ob protein. The levels of expression were estimated 
to be &gt;200 ng/10 .mu.g total protein, or human ob expression is &gt;2% of 
total cell protein. On non-reducing gels, the ob protein appears as a 
doublet with equal amounts of protein in each band of the doublet. This 
doublet collapses to a single band upon reduction, suggesting that the 
protein derived from cell lysates contains disulfide bonded and reduced 
material. Roughly 50% of the recombinant human ob protein pellets upon 
centrifugation at 20,000.times.g. The transformants produced roughly 40 
mg/L of human ob in shake flask culture. 
Transformant CZY112 strains were found to mainly produce uncleaved, 
glycosylated .alpha.-Factor prepro:ob fusion protein, most of which was 
secreted from the cell and present in the cell media. Small amounts of 
cleaved ob with a molecular weight expected for the authentic product were 
produced, and most of which was retained in an intracellular compartment. 
Based on dilution analysis and Western blotting, there is about 2 mg/L of 
cleaved ob is intracellular and about 1 mg/L of cleaved ob, is found in 
the medium of CZY112 cells grown in shake flask. 
Transformant CZY113 strains were found to produce high levels of cleaved ob 
protein and low levels of a protein of the size expected for an 
.alpha.-Factor prepro:ob fusion protein. In addition, the overall level of 
ob expression was elevated significantly. Coomasie staining and Western 
blotting revealed these cells produced a cleaved, intracellular form of 
human ob that was about 1% of total cell protein. The secreted levels of 
cleaved ob were about 5-fold lower. Gel analysis demonstrated that this 
form of ob migrated slightly slower than either the cytoplasmic form or 
the CZY112 produced form of ob. Most of the intracellular form was in the 
soluble phase of a sample spun at 20,000.times.g, suggesting it was not 
aggregated. The level of expression of cleaved human ob protein from 
CZY113 strains grown in shake flask were estimated to be 20 mg/L for the 
intracellular form and 4 mg/L for the secreted form. 
Example 15 
Distinctions Between ob Protein and ACM 
A. Heat Inactivation 
A preparation of Zucker rat adipose conditioned medium (ACM) was prepared 
as in Example 2.A., above. When tested in the ob/ob mouse bioassay, mice 
(n=3) injected with this ACM preparation demonstrated appetite suppression 
that was 49.+-.8.6% of baseline 24 h chow intake. When this same ACM 
preparation was heated to 95.degree. C. for 5 min, mice injected with this 
heated ACM preparation demonstrated appetite suppression that was 
94.+-.4.2% of baseline 24 h chow intake. Therefore, the active appetite 
suppression factor(s) in ACM were inactivated after heating at 95.degree. 
C. for 5 min. 
In contrast, conditioned medium containing recombinant ob protein was heat 
stable. Briefly, medium conditioned by BHK cells expressing recombinant ob 
protein was maintained at 4.degree. C. or was heated at 95.degree. C. for 
10-20 min. Each preparation was tested in the mouse bioassay by daily 
intraperitoneal injections over a two week period (n=3 for each 
preparation tested). Each preparation of BHK/ob conditioned medium 
produced significant and sustained reductions in body weight and chow 
intake. The heated preparation retained approximately 70% of the appetite 
suppresive activity of the unheated preparation. 
B. HPLC Analysis 
Adipose-conditioned medium was subjected to HPLC and analyzed for ob 
protein. Briefly, a preparation of Zucker rat ACM was prepared as in 
Example 2.A., above. When tested in the ob/ob mouse bioassay, the mice 
injected with this ACM preparation demonstrated appetite suppression that 
was 61.+-.11% of baseline chow intake. A 50 .mu.l sample of this ACM 
preparation was injected onto a Vydac C4 HPLC column that was equilibrated 
in 27% acetonitrile/0.1% trifluoroacetic acid (TFA). The HPLC column was 
eluted using a 60 min gradient from 27% acetonitrile/0.1% TFA to 80% 
acetonitrile/0.1% TFA at a flow rate of 1 ml/min. Ten min fractions were 
collected and monitored at 215 nm, 0.2 OD full scale. The fractions were 
lyophilized and redissolved in 30 .mu.l SDS-PAGE sample loading buffer 
(see Example 2.D.). A 15 .mu.l aliquot of each of redissolved fractions 
21-40 were electrophoresed using 10-20% Tris/Tricine gels. The gels were 
immunoblotted (see Example 11(2)) using an affinity-purified rabbit 
anti-mouse ob antibody preparation (see Example 10.B.). 
Zucker rat adipose conditioned medium contained a fraction reactive with 
anti-ob antibodies as determined by Western blotting. This fraction eluted 
from the HPLC column between 31.6 and 32.6 min. This retention profile was 
very similar to that observed by equivalent HPLC analysis of recombinant 
mouse ob protein expressed in BHK cells (i.e., between 31.9 and 32.9 min). 
The retention time for purified, recombinant mouse ob protein that was 
expressed in BHK cells was 31.99 min. The estimated concentration of rat 
ob protein in this peak is .ltoreq.16 .mu.g/ml, as determined by 
comparison to a purified mouse ob standard of known protein concentration. 
Upon further analysis of additional rat ACM HPLC fractions, one band 
detected by immunoblotting had a retention time between 20.6 and 21.6 min, 
and an apparent molecular weight between 43 kD (ovalbumin) and 68 kD 
(BSA). 
C. Specific Activity 
The specific activity of recombinant ob protein and ACM preparations was 
determined. Briefly, a 35.times. concentrate of medium conditioned by BHK 
cells expressing mouse ob protein was tested in the mouse bioassay and 
examined by immunoblotting. By mouse bioassay, this BHK/ob preparation 
demonstrated appetite suppression that was 71.+-.10% of baseline 24 h chow 
intake. For immunoblotting, dilutions of a purified recombinant ob protein 
standard (expressed in E. coli; having a known protein concentration) were 
electrophoresed on the same gel with dilutions of the 35.times. BHK/ob 
concentrated medium. Bands corresponding to ob protein were detected using 
affinity-purified rabbit anti-mouse ob antibody. A 10 .mu.l aliquot of a 
1:80 dilution of 35.times. BHK/ob concentrate and a 10 ng ob protein 
standard yielded bands of similar intensity. The 35.times. concentrate was 
calculated to contain 80 .mu.g/ml recombinant ob protein (80.times.10 ng 
of ob protein=800 ng.div.10 .mu.l of BHK/ob concentrate=80 .mu.g/ml). The 
protein concentration of the same conditioned medium preparation before 
concentration was estimated to be about 1-2 .mu.g/ml. 
Zucker fat ACM was similarly assayed. By mouse bioassay, two distinct ACM 
preparations demonstrated appetite suppression that ranged from 17.+-.5% 
to 57.+-.13% of baseline 24 h chow intake. A 7.5 .mu.l aliquot of a 
28.times. concentrate of ACM obtained using adipose tissue from a young 
rat, and a 7.5 .mu.l aliquot of a 50.times. concentrate of ACM obtained 
using adipose tissue from an old rat were electrophoresed with dilutions 
of the ob protein standard, and with an aliquot of 74.times. concentrated 
BHK/ob conditioned medium (appetite suppression activity=83.+-.15% of BL 
For both concentrated ACM aliquots, less than 5 ng -ob protein was 
detected. In contrast, the concentrated BHK/ob aliquot contained &gt;50 ng ob 
protein. 
Preparations of BHK/ob conditioned medium and Zucker ACM were 
electrophoresed and silver stained, and relative amounts of protein in the 
predicted ob protein band were determined. Briefly, a 15.times. BHK/ob 
concentrate and a 20.times. concentrated ACM preparation were 
electrophoresed as described in Example 11. above. In the BHK/ob 
concentrate, a stained band was visible at the predicted ob protein 
location; no band was detected in the ACM preparation. Immunoblotting of 
the same preparations was performed. In the BHK/ob concentrate, ob protein 
was detected. In contrast, ob protein was only detected in the ACM 
preparation after a very extensive exposure period. 
Taken together, these data demonstrate that significant appetite 
suppressive activity was present in ACM preparations that contained very 
low amounts of detectable ob protein. In a similarly concentrated BHK/ob 
preparation exhibiting less appetite suppression activity than the ACMs, 
at least 10.times. more ob protein was detected. These data suggest that 
additional factors or cofactors present in the ACMs (i) have appetite 
suppressive activity, or (ii) potentiate the activity of ob protein 
present in ACMs. Alternatively, these results suggest that ob protein 
produced and secreted in a natural adipose tissue environment is different 
from, and more active than, recombinant ob protein expressed in a 
heterologous host cell. These alternative explanations are not mutually 
exclusive. 
D. Expected Endocrine Levels of ob Protein 
In the mouse bioassay system, a significant, reproducible decrease in 24 h 
chow intake was observed with single, daily intraperitoneal injections of 
50-100 .mu.g recombinant ob protein. Since the recipient mice weigh 
approximately 50 g, the effective appetite suppressive dose in this system 
is in the range of about 1-2 mg/kg body weight. 
In contrast, effective daily doses of insulin and glucagon in humans are in 
the range of about 0.01-0.02 mg/kg body weight. The relatively low potency 
of recombinant ob protein (2 logs less than the hormones insulin and 
glucagon) suggests that an additional factor or cofactor, or an in vivo 
modification, may be necessary to obtain a purified recombinant ob protein 
preparation having enhanced potentcy in vivo. These data are consistent 
with the results obtained in Section C., above. 
Example 16 
ELISA for Detection of Circulating ob Protein in Human Serum 
The secretion of ob protein is predicted to be a critical mechanism by 
which adipocytes "signal" the total body fat mass to the central nervous 
system (CNS). This signal then plays a role in regulating energy balance 
in an individual mammal. The condition of obesity may result from (i) an 
inadequate level of or a mutated circulating ob protein; (ii) an absent or 
defective receptor for ob protein; and/or (iii) a defect in the ob-CNS 
signaling pathway. An ELISA that detects levels of circulating ob protein 
in human serum provided further data on the relationship of ob protein and 
obesity. 
A. ELISA 
Mouse monoclonal antibodies directed against human ob protein (HOB) were 
generated, as described in Example 10.B. Briefly, mice were immunized with 
250 .mu.g native MBP-HOB fusion protein, and were boosted three times with 
125 .mu.g native MBP-HOB. Hybridomas were prepared using immune spleen 
cells and an SP2/0-E fusion partner. Three monoclonal antibodies 
216.1.2.1.2; 216.3.3.2.1; 218.5.4.2) were subjected to further analysis. 
The following format was used in a preliminary ELISA for detection of human 
ob protein in human serum: 
(a) Microtiter plates were coated with goat anti-mouse IgG, and the wells 
were blocked and washed; 
(b) A hybridoma supernatant that contained anti-HOB antibody was incubated, 
and the wells were washed; 
(c) Serum samples containing known amounts of exogenously added HOB 
("spiked with HOB"; 0 to 100 ng HOB/ml serum diluent) were incubated, and 
the wells were washed; 
(d) Affinity-purified rabbit anti-HOB polyclonal antibody was added (1:1000 
dilution) and incubated, and the wells were washed; 
(e) Goat anti-rabbit IgG conjugated to horseradish peroxidase (GAR-HRP; 
Biosource, Camarillo, Calif.) was added (1:2000 dilution) and incubated, 
and the wells were washed; 
(f) ortho-phenylenediamine dihydrochloride (OPD; Sigma) was added and 
incubated, and color development was stopped with 1N H.sub.2 SO.sub.4 ; 
and 
(g) The contents of the wells were read at 490 nm. 
Serum diluents used in (c), above, included RPMI-serum-free (SF) (culture 
medium RPMI-1640; JRH); RPMI+15% fetal calf serum (FCS); 100% ob/ob mouse 
serum; 100% normal human serum; 50% normal human serum (diluted in 
RPMI-SF). Controls included unspiked diluent blanks (RPMI-SF and RPMI-15% 
FCS). The recombinant HOB used to generate a standard curve (for spiking) 
was produced in yeast. The rabbit anti-HOB serum was raised to recombinant 
HOB produced in E. coli. 
The presence of 100% mouse or human serum inhibited and/or interfered with 
the detection of spiked HOB. For instance, in samples spiked with 12.5 
ng/ml HOB, the following values were obtained: RPMI-SF=1.308; RPMI-15% 
FCS=1.6561 100% mouse serum=0.203; 100% human serum=0.326; 50% human 
serum=0.407. In addition, the background values of the unspiked diluents 
varied among samples (RPMI-SF=0.470; RPMI-15% FCS=0.467). In the presence 
of 50% or 100% unspiked normal human serum, the background values were 
0.186 and 0.177, respectively. 
The ELISA format was modified as follows: 
(1) Instead of steps (a) and (b), above, microtiter wells were coated with 
one of three purified mouse monoclonal antibodies. 
(2) In step (c), the serum content was diluted to 10% (by diluting an 
HOB-spiked 100% serum sample to 10% serum, or by spiking HOB into 10%, 
rather than 100%, human serum--equivalent results were obtained using 
either 
Using serum samples spiked with HOB to yield a final concentration of 12.5 
ng HOB/ml sample, and using monoclonal antibody 216.3.3.2.1 as the capture 
antibody, the following representative results were obtained: 100% human 
serum=0.239; 50% human serum=0.582; 10% human serum=0.936; buffer 
(containing salts and BSA)=1.654. At 10% human serum, A.sub.490 values 
ranged from 2.831 (200 ng HOB/ml) to 0.432 (3.13 ng HOB/ml). 
The capture monoclonal antibody concentration was optimized using a spike 
concentration of 5 ng HOB/ml. Three purified monoclonal antibodies were 
diluted (from 2.5 .mu.g/ml to 1.22 ng/ml), and 100 .mu.l/well was added to 
each microtiter well. The monoclonals were then incubated for 2 h at 
37.degree. C. The capture antibody was incubated with 10% normal human 
serum alone or with 10% normal human serum spiked with 5 ng/ml ob protein 
for 2 h at 37.degree. C. Four dilutions (1:1000; 1:3500; 1:7000 and 
1:10,500) of detecting antibody, rabbit anti-HOB, were added and incubated 
for 1 h at 37.degree. C. The best signal-to-noise ratio (ob protein-spiked 
vs. unspiked 10% human serum; ratio=7.6) was achieved with capture 
monoclonal antibody 216.3.3.2.1 at 2500 ng/ml and rabbit anti-HOB at 
1:1000. 
To confirm specificity of the assay, some standard curve and patient 
samples were preincubated with an excess of free monoclonal antibody (50 
.mu.g/ml) for 30 min at RT, and were compared to analogous standard curve 
and patient samples that were incubated without added free monoclonal 
antibody. In the samples that were preincubated with an excess of 
monoclonal antibody, the signal was signficantly reduced, indicating the 
ob protein specificity of this ELISA. 
B. Testing of Human Serum Samples by ELISA 
The ELISA used for testing human serum samples was performed as follows: 
(a) Microtiter plates were coated with purified capture monoclonal antibody 
(216.1.2.1.2 or 216.3.3.2.1; added at 2.5 .mu.g/ml and incubated overnight 
at 4.degree. C.), and the wells were blocked with 1% BSA for 2 h at 
37.degree. C., and washed (four times with PBS, pH 7.2+TWEEN-20 5 ml/10 
l! at 250 .mu.l/well); 
(b) Spiked (standard curve) samples (prepared in buffered RPMI-SF or in 
buffer containing salts and 1% BSA) or human serum samples (diluted to 10% 
in RPMI-SF) were added to wells and incubated for 2.5 h at 37.degree. C., 
and the wells were washed as in (a); 
(c) Affinity-purified rabbit anti-HOB polyclonal antibody was added (1:500 
dilution in 1% BSA) and incubated for 1 h at 37.degree. C., and the wells 
were washed as in (a); 
(d) Goat anti-rabbit IgG conjugated to horseradish peroxidase (GAR-HRP) was 
added (1:2000 dilution in 1% BSA) and incubated for 1 h at 37.degree. C., 
and the wells were washed as in (a); 
(e) Ortho-phenylenediamine dihydrochloride (OPD; Sigma; prepared at 0.4 
mg/ml in 63 mM sodium citrate and 37 mM citric acid, pH 5) was added (100 
.mu.l/well) and incubated 7 min at RT, and color development was stopped 
with 1N H.sub.2 SO.sub.4 (100 .mu.l/well); 
(f) The contents of the wells were read at 490 nm using an automated 
microtiter plate reader. 
The following results were obtained using ELISA and human serum samples 
(100 .mu.l/well; triplicate determinations) containing unknown quantities 
of circulating ob protein: 
______________________________________ 
INDIVIDUAL SERUM SAMPLES 
MAb 216.1.2.1.2 
MAb 216.3.3.2.1 
Human Sample Average A490 Value 
______________________________________ 
JC37 0.214 0.221 
S063 0.217 0.229 
JJ11 0.194 0.214 
SR30 0.144 0.154 
FC23 0.301 0.303 
CB46 0.155 0.172 
MC10 0.497 0.593 
SB62 0.397 0.48 
______________________________________ 
STANDARD CURVE 
MAb 216.1.2.1.2 
MAb 216.3.3.2.1 
Spiked 10% 
Human serum-RPMI-SF 
Average A490 Value 
______________________________________ 
200 ng HOB/ml 2.935 2.733 
100 ng HOB/ml 2.8 2.671 
50 ng HOB/ml 2.4 2.197 
25 ng HOB/ml 1.61 1.565 
12.5 ng HOB/ml 0.937 0.899 
6.25 ng HOB/ml 0.526 0.574 
3.13 ng HOB/ml 0.385 0.38 
1.56 ng HOB/ml 0.271 0.279 
0.78 ng HOB/ml 0.231 0.218 
0.39 ng HOB/ml 0.211 0.197 
0.20 ng HOB/ml 0.203 0.195 
______________________________________ 
______________________________________ 
STANDARD CURVE 
Spiked 10% MAb 216.1.2.1.2 
MAb 216.3.3.2.1 
Hu serum-RPMI-SF 
Average A490 Value 
______________________________________ 
200 ng HOB/ml 2.935 2.733 
100 ng HOB/ml 2.8 2.671 
50 ng HOB/ml 2.4 2.197 
25 ng HOB/ml 1.61 1.565 
12.5 ng HOB/ml 
0.937 0.899 
6.25 ng HOB/ml 
0.526 0.574 
3.13 ng HOB/ml 
0.385 0.38 
1.56 ng HOB/ml 
0.271 0.279 
0.78 ng HOB/ml 
0.231 0.218 
0.39 ng HOB/ml 
0.211 0.197 
0.20 ng HOB/ml 
0.203 0.195 
______________________________________ 
Using yeast produced recombinant ob protein as a standard, the detection 
limit of the assay performed in 10% human serum was about 3 ng/ml. 
Certain data were obtained relating to each of the individuals whose serum 
was tested above: sex, age, height in cm, weight in kg, body mass index, 
percent body fat, smoker/not, diabetic/not and onset of obesity. Body mass 
index (BMI) is defined as weight in kg.div.height in m.sup.2. A person 
with a BMI&gt;28% is considered to be overweight. Body fat content (%BF) was 
measured after a 6 h fast and after a 24 h period of avoiding 
gas-producing foods and liquids (i.e., carbonated beverages). Each 
individual's residual lung volume was determined, and this value was 
subtracted from immersion (underwater) weight in a bathing suit, so that 
only an individual's mass, and not air, was measured. When data obtained 
from 40 normal and obese subjects were subjected to linear regression 
analysis, a strong correlation between BMI and % BF was found (0.718). 
These 40 subjects did not have a history of chronic heart or chronic lung 
disease, and were not limiting caloric intake (i.e., dieting) at the time 
of the study. 
Eight subjects at the upper and lower extremes of BMI and/or % BF, selected 
for ELISA determination, above, are profiled below: 
______________________________________ 
Onset 
Di- Obe- 
Subj Sex Age Ht Wt BMI % BF Smoke ab sity 
______________________________________ 
JC37 M 60 187 122 35.13 
37.2 N Y* Adult 
SO63 M 39 208 166 -- -- N Y* Child 
JJ11 M 45 178 85 27.11 
14.7 N N N/A 
SR30 F 46 157 55 22.46 
14.9 N N N/A 
FC23 M 62 184 69 20.38 
11.8 N N N/A 
CB46 M 26 197 75 19.27 
10.5 N N N/A 
MC10 F 40 159 123 48.57 
52 N N Adult 
SB62 F 33 181 83 25.44 
32.9 N N N/A 
______________________________________ 
*These subjects were diagnosed with Type II diabetes. 
JC37 was on medication; SO63 was not. 
The three obese subjects had an average body fat content of 47.8.+-.2.3%; 
the five control subjects had an average body fat content of 19.3.+-.4.6%. 
The serum HOB level (determined by multiplying the HOB value in 10% serum 
by 10) in the three obese subjects averaged 43.2.+-.19.4 ng/ml; the five 
control subjects averaged 6.8.+-.3.2 ng/ml (p=0.049). These results 
indicate that obese subjects do not have deficient levels of circulating 
ob protein; in fact, the three obese subjects tested exhibited circulating 
ob levels that were 6 times higher than those of five normal subjects. 
From the foregoing, it will be appreciated that, although specific 
embodiments of the invention have been described herein for purposes of 
illustration, various modifications may be made without deviating from the 
spirit and scope of the invention. Accordingly, the invention is not 
limited except as by the appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 10 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 504 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..501 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATGTGCTGGAGACCCCTGTGTCGGTTCCTGTGGCTTTGGTCCTATCTG48 
MetCysTrpArgProLeuCysArgPheLeuTrpLeuTrpSerTyrLeu 
151015 
TCTTATGTTCAAGCAGTGCCTATCCAGAAAGTCCAGGATGACACCAAA96 
SerTyrValGlnAlaValProIleGlnLysValGlnAspAspThrLys 
202530 
ACCCTCATCAAGACCATTGTCACCAGGATCAATGACATTTCACACACG144 
ThrLeuIleLysThrIleValThrArgIleAsnAspIleSerHisThr 
354045 
CAGTCGGTATCCGCCAAGCAGAGGGTCACTGGCTTGGACTTCATTCCT192 
GlnSerValSerAlaLysGlnArgValThrGlyLeuAspPheIlePro 
505560 
GGGCTTCACCCCATTCTGAGTTTGTCCAAGATGGACCAGACTCTGGCA240 
GlyLeuHisProIleLeuSerLeuSerLysMetAspGlnThrLeuAla 
65707580 
GTCTATCAACAGGTCCTCACCAGCCTGCCTTCCCAAAATGTGCTGCAG288 
ValTyrGlnGlnValLeuThrSerLeuProSerGlnAsnValLeuGln 
859095 
ATAGCCAATGACCTGGAGAATCTCCGAGACCTCCTCCATCTGCTGGCC336 
IleAlaAsnAspLeuGluAsnLeuArgAspLeuLeuHisLeuLeuAla 
100105110 
TTCTCCAAGAGCTGCTCCCTGCCTCAGACCAGTGGCCTGCAGAAGCCA384 
PheSerLysSerCysSerLeuProGlnThrSerGlyLeuGlnLysPro 
115120125 
GAGAGCCTGGATGGCGTCCTGGAAGCCTCACTCTACTCCACAGAGGTG432 
GluSerLeuAspGlyValLeuGluAlaSerLeuTyrSerThrGluVal 
130135140 
GTGGCTTTGAGCAGGCTGCAGGGCTCTCTGCAGGACATTCTTCAACAG480 
ValAlaLeuSerArgLeuGlnGlySerLeuGlnAspIleLeuGlnGln 
145150155160 
TTGGATGTTAGCCCTGAATGCTGA504 
LeuAspValSerProGluCys 
165 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 167 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetCysTrpArgProLeuCysArgPheLeuTrpLeuTrpSerTyrLeu 
151015 
SerTyrValGlnAlaValProIleGlnLysValGlnAspAspThrLys 
202530 
ThrLeuIleLysThrIleValThrArgIleAsnAspIleSerHisThr 
354045 
GlnSerValSerAlaLysGlnArgValThrGlyLeuAspPheIlePro 
505560 
GlyLeuHisProIleLeuSerLeuSerLysMetAspGlnThrLeuAla 
65707580 
ValTyrGlnGlnValLeuThrSerLeuProSerGlnAsnValLeuGln 
859095 
IleAlaAsnAspLeuGluAsnLeuArgAspLeuLeuHisLeuLeuAla 
100105110 
PheSerLysSerCysSerLeuProGlnThrSerGlyLeuGlnLysPro 
115120125 
GluSerLeuAspGlyValLeuGluAlaSerLeuTyrSerThrGluVal 
130135140 
ValAlaLeuSerArgLeuGlnGlySerLeuGlnAspIleLeuGlnGln 
145150155160 
LeuAspValSerProGluCys 
165 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 966 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 46..546 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GCGCCAGCGGTTGCAAGGCCCAAGAAGCCCATCCTGGGAAGGAAAATGCATTGG54 
MetHisTrp 
GGAACCCTGTGCGGATTCTTGTGGCTTTGGCCCTATCTTTTCTATGTC102 
GlyThrLeuCysGlyPheLeuTrpLeuTrpProTyrLeuPheTyrVal 
51015 
CAAGCTGTGCCCATCCAAAAAGTCCAAGATGACACCAAAACCCTCATC150 
GlnAlaValProIleGlnLysValGlnAspAspThrLysThrLeuIle 
20253035 
AAGACAATTGTCACCAGGATCAATGACATTTCACACACGCAGTCAGTC198 
LysThrIleValThrArgIleAsnAspIleSerHisThrGlnSerVal 
404550 
TCCTCCAAACAGAAAGTCACCGGTTTGGACTTCATTCCTGGGCTCCAC246 
SerSerLysGlnLysValThrGlyLeuAspPheIleProGlyLeuHis 
556065 
CCCATCCTGACCTTATCCAAGATGGACCAGACACTGGCAGTCTACCAA294 
ProIleLeuThrLeuSerLysMetAspGlnThrLeuAlaValTyrGln 
707580 
CAGATCCTCACCAGTATGCCTTCCAGAAACGTGATCCAAATATCCAAC342 
GlnIleLeuThrSerMetProSerArgAsnValIleGlnIleSerAsn 
859095 
GACCTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCTCTAAG390 
AspLeuGluAsnLeuArgAspLeuLeuHisValLeuAlaPheSerLys 
100105110115 
AGCTGCCACTTGCCCTGGGCCAGTGGCCTGGAGACCTTGGACAGCCTG438 
SerCysHisLeuProTrpAlaSerGlyLeuGluThrLeuAspSerLeu 
120125130 
GGGGGTGTCCTGGAAGCTTCAGGCTACTCCACAGAGGTGGTGGCCCTG486 
GlyGlyValLeuGluAlaSerGlyTyrSerThrGluValValAlaLeu 
135140145 
AGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAGCTGGACCTC534 
SerArgLeuGlnGlySerLeuGlnAspMetLeuTrpGlnLeuAspLeu 
150155160 
AGCCCTGGGTGCTGAGGCCTTGAAGGTCACTCTTCCTGCAAGGACTACGTTA586 
SerProGlyCys 
165 
AGGGAAGGAACTCTGGCTTCCAGGTATCTCCAGGATTGAAGAGCATTGCATGGACACCCC646 
TTATCCAGGACTCTGTCAATTTCCCTGACTCCTCTAAGCCACTCTTCCAAAGGCATAAGA706 
CCCTAAGCCTCCTTTTGCTTGAAACCAAAGATATATACACAGGATCCTATTCTCACCAGG766 
AAGGGGGTCCACCCAGCAAAGAGTGGGCTGCATCTGGGATTCCCACCAAGGTCTTCAGCC826 
ATCAACAAGAGTTGTCTTGTCCCCTCTTGACCCATCTCCCCCTCACTGAATGCCTCAATG886 
TGACCAGGGGTGATTTCAGAGAGGGCAGAGGGGTAGGCAGAGCCTTTGGATGACCAGAAC946 
AAGGTTCCCTCTGAGAATTC966 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 167 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetHisTrpGlyThrLeuCysGlyPheLeuTrpLeuTrpProTyrLeu 
151015 
PheTyrValGlnAlaValProIleGlnLysValGlnAspAspThrLys 
202530 
ThrLeuIleLysThrIleValThrArgIleAsnAspIleSerHisThr 
354045 
GlnSerValSerSerLysGlnLysValThrGlyLeuAspPheIlePro 
505560 
GlyLeuHisProIleLeuThrLeuSerLysMetAspGlnThrLeuAla 
65707580 
ValTyrGlnGlnIleLeuThrSerMetProSerArgAsnValIleGln 
859095 
IleSerAsnAspLeuGluAsnLeuArgAspLeuLeuHisValLeuAla 
100105110 
PheSerLysSerCysHisLeuProTrpAlaSerGlyLeuGluThrLeu 
115120125 
AspSerLeuGlyGlyValLeuGluAlaSerGlyTyrSerThrGluVal 
130135140 
ValAlaLeuSerArgLeuGlnGlySerLeuGlnAspMetLeuTrpGln 
145150155160 
LeuAspLeuSerProGlyCys 
165 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 438 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..435 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GTCCCTATCCAGAAAGTCCAGGATGACACCAAAACCCTCATCAAGACC48 
ValProIleGlnLysValGlnAspAspThrLysThrLeuIleLysThr 
151015 
ATTGTCACCAGGATCAATGACATTTCACACACGTCGGTATCCGCCAAG96 
IleValThrArgIleAsnAspIleSerHisThrSerValSerAlaLys 
202530 
CAGAGGGTCACTGGCTTGGACTTCATTCCTGGGCTTCACCCCATTCTG144 
GlnArgValThrGlyLeuAspPheIleProGlyLeuHisProIleLeu 
354045 
AGTTTGTCCAAGATGGACCAGACTCTGGCAGTCTATCAACAGGTCCTC192 
SerLeuSerLysMetAspGlnThrLeuAlaValTyrGlnGlnValLeu 
505560 
ACCAGCCTGCCTTCCCAAAATGTGCTGCAGATAGCCAATGACCTGGAG240 
ThrSerLeuProSerGlnAsnValLeuGlnIleAlaAsnAspLeuGlu 
65707580 
AATCTCCGAGACCTCCTCCATCTGCTGGCCTTCTCCAAGAGCTGCTCC288 
AsnLeuArgAspLeuLeuHisLeuLeuAlaPheSerLysSerCysSer 
859095 
CTGCCTCAGACCAGTGGCCTGCAGAAGCCAGAGAGCCTGGATGGCGTC336 
LeuProGlnThrSerGlyLeuGlnLysProGluSerLeuAspGlyVal 
100105110 
CTGGAAGCCTCACTCTACTCCACAGAGGTGGTGGCTTTGAGCAGGCTG384 
LeuGluAlaSerLeuTyrSerThrGluValValAlaLeuSerArgLeu 
115120125 
CAGGGCTCTCTGCAGGACATTCTTCAACAGTTGGATGTTAGCCCTGAA432 
GlnGlySerLeuGlnAspIleLeuGlnGlnLeuAspValSerProGlu 
130135140 
TGCTGA438 
Cys 
145 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 145 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ValProIleGlnLysValGlnAspAspThrLysThrLeuIleLysThr 
151015 
IleValThrArgIleAsnAspIleSerHisThrSerValSerAlaLys 
202530 
GlnArgValThrGlyLeuAspPheIleProGlyLeuHisProIleLeu 
354045 
SerLeuSerLysMetAspGlnThrLeuAlaValTyrGlnGlnValLeu 
505560 
ThrSerLeuProSerGlnAsnValLeuGlnIleAlaAsnAspLeuGlu 
65707580 
AsnLeuArgAspLeuLeuHisLeuLeuAlaPheSerLysSerCysSer 
859095 
LeuProGlnThrSerGlyLeuGlnLysProGluSerLeuAspGlyVal 
100105110 
LeuGluAlaSerLeuTyrSerThrGluValValAlaLeuSerArgLeu 
115120125 
GlnGlySerLeuGlnAspIleLeuGlnGlnLeuAspValSerProGlu 
130135140 
Cys 
145 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
TAAAAAGAATTCAAAAATGGTGCCCATCCAAAAAGTCCAA40 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
ACGACTGTCGACTCAGCACCCAGGGCTGAG30 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 43 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GAACAGAAGCTTGGACAAGAGAGTGCCCATCCAAAAAGTCCAA43 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 49 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GAACAGAAGCTTGGACAAGAGAGAAGCTGTGCCCATCCAAAAAGTCCAA49 
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