Note: Descriptions are shown in the official language in which they were submitted.
21 90609
WO 95/32727 I ~ '103~
INHIBITION OF INFECTION OF MAMMALIAN CELLS BY RESPIRATORY SYNCYTIAL VIRUS
The present i nventi on rel ates genera l l y to i nh i bi ti ng the i nfecti onof mammalian cells by Respiratory Syncytial Virus, and more specifically to
the use of native or recombinant human ,B-casein and hydrolysates thereof for
inhibiting the infection of mammalian cells by Respiratory Syncytial Virus.
Respiratory Syncytial Virus ~RSV) is the single most frequent cause
of acute respiratory tract infection in infants and children. Infants less
than six months of age are most frequently and seriously affected. In most
immunologically normal subjects, infection with RSV is limited to the
respi ratory mucosa, and i s associ ated wl th the devel opment of bronchi ol i ti s,
pneumonia and reactive airway disease. RSV infection in immunocompromised
subjects has until recently been associated with increased mortality in
infants and increased morbidity in other age groups. It has recently been
reported in PEnIATRIC NOTES, Vol. 18, No. 14. January 27, 1994, that periods
of high incidence of acute respiratory disease and numbers of deaths in
elderly people were followed within 2-3 weeks by reports of high numbers of
RSV or influenza virus isolates. The analyses indicate that RSV is as
important as influenza viruses in causing morbidity and deaths among the
el derly .
It has been reported that some respi ratory di sease may be prevented
by breast feeding, and that "bronchiolitis of infants due to respiratory
syncytial virus is less frequent in breast fed than in artificially fed
infants". While human breast milk can contain antibodies to RSV, it has
been found that milk also has antiviral activity that is not due to
anti bodi es . It has been theori zed that thi s effect "may be produced by
certain polysaccharides which are found on a number of different molecular
constituents of milk." Tyrrell, "BREAST FEEDING AND VIRUS INFECTIONS", THE
IMMUNOLOGY OF INFANT FEEDING, edited by A. W. Wilkinson, Plenum Press, New
York, NY pages 55-62 (1981).
Okamato, et a l . . "Anti vi ral Factors i n Human Mi l k: Impl i cati ons i n
Respiratory Syncytial Virus Infection", ACTA PAEDIATRICA SCANDANAVICA
SUPPLEMENT, 351:137-143 (1989) disclose that while ,the mechanisms of
protective immunity to RSV had not been clearly defined, immunity acqu~red
WO 95132727 2 1 9 0 6 0 9 PCTII~S9~/03628
transplacentally or via breast feeding has been suggested to reduce the risk
of lower respiratory tract disease. However, this publication focuses upon
the role of antibodies transmitted in breast milk or the possible role of
breast milk in modulating an infant's RSV immune response.
Laegreid et al.. "Neutralizing Activity in Human Milk Fractions
against Respiratory Syncytial Virus", ACTA PAEDIATRICA SCANDANAVICA, 75:6g6-
701 (1986) reports a study which confirms that human milk may contain RSV-
neutralizing activity b~a non-immunoglobulin nature as well as RSV-specific
antibody. However, the identity and mechanism of the non-immunoglobulin
anti-RSV component of human milk is not identified. It is important though
to note that Laegreid et al. disclose that RSV-neutralizlng components from
breast milk may reach an infant's respiratory tract directly as a result of
regurgitation and inhalation of milk during and after feeding. The mucosa
of the respiratory tract may gain direct protection in this way.
WO 91/06308 filed by Andersson et al . for "ANTIBACTERIAL COMPOSITION",
and a published article by the same authors (Aniansson et al., "Anti-
adhesive activity of human casein against Streptococcus pneumonia and
Haemophilus influenzae", MICROBIAL PATHOGENESIS, 8:315-323 (1990) disclose
the use of a milk fraction having a molecular weight of at least 5~000
daltons for "therapeutic prophylactic, and/or diagnostic use in infections
caused by 5. pneumonae and/or H. inf7uenz~e" but it is suggested in these
publications that the beneficial effect is provided by kappa-casein.
However, the present invention relates to the use of native or recombinant
human ~-casein and hydrolysates of both to inhibit RSV infections.
WO93/04172 relates to a DNA sequence encoding human ~-casein, but does
not disclose the capacity of either native or recombinant human ,~-casein to
inhibit the attachment of RSV to human cells.
IIO91/08675 discloses an infant formula which contains recombinant
forms of both human alpha-lactalbumin and human ~-casein. However, this
publication discloses only that these human milk proteins will "give a
simulated human mother's milk formula that does not exhibit the allergenic
properties associated with formulas based on cow or other foreign protein.~
(page 3, lines 20-22). The use of human ,B-casein to inhibit the attachment
of RSV to human cells is not taught or suggested ln said publication.
The two assays (a HEp-2 cell assay and a LLC-MK2 cell assay) which
were used for determining the bioactivity of ,~-casein are described below.
These assays have not been published heretofore, although the HEp-2 cell
WO9513t727 2 i 9060~ P~
assay was based upon established methodology.
MATERIALS USED IN BOTH ASSAYS
Native Human B-Casein
B-casein isolated from human milk was purchased from Symbicom AB, P.O.
Box 1451, S-9OI 24 Umea, Sweden.
Recombinant Human B-Casein
Applicants obtained B-casein cDNA and the expression system from
Symbicom AB, P.O. Box 1451, S-901 24 Umea, Sweden. The human B-casein cDNA
used had been previously cloned and sequenced by Lonnerdal et al., Cloning
and sequencing of a cDNA encoding human milk B-casein. (SEQ.ID NO: 1:)
Federation of Europedn Biochen1ica7 Societies Letters 269, 153-156 (1990).
The recombinant human ~7-casein was obtained from E. co1i and purified
according to the method of Hansson et al., "Expression of Human Milk 6-
Casein in Escherichia co1i: Comparison of Recombinant Protein with Native
Isoforms". Protein Expression and Purification 4, 373-381 (1993). To
express human B-casein in E.co1i, B-casein cDNA was cloned under control of
a T7 promoter in two different expression vectors. One vector, pS26, was
designed for intracellular expression. The other vector, pS28, has a signal
sequence for extracellular expression. The procedure followed was
substantially that described by Hansson et al.
Human B-casein cDNA was isolated by Hansson et al. as a 1.1-kb EcoRI
fragment from a human lambda gt mammary gland library, and was subcloned
into pUC19, which was designated pS21. The cDNA was modified by
introduction of synthetic oligonucleotides in the 5' and 3' termini. To
introduce a suitable cloning site in the 5' end, Ndel, a translational
start, was inserted in front of the sequence encoding mature human B-casein.
To adapt the initial part of the translated sequence to E.co1i codon usage,
six synthetic oligonucleotides were constructed and ligated. Also, PstI and
EcoRI sites were inserted in front of the NdeI site. The sequence of the
synthetic fragment was 5'-CTGCAGAATTCATATGCGT
GMMCCATCGMTCCCTGAGCTCGAGCGMGMTCGATCACCGMTACAAAAAAGTTGMAMGTTMMCACG
AGGACCAGGATCC-3'. (SEQ ID NO: 2:) The protein encoding sequence is
underlined. The synthetic fragment was cloned into PstI/Ban1HI-digested
pUC19 resulting in plasmid pS24. To insert the rest of the B-casein
woss/32727 2 ~ 9 06 09 P~
encoding sequence, a 303-bp AccI/Bg1II fragment was isolated and cloned into
a pUC18 derivative and designated plasmid pS22. Four synthetic
oligonucleotides containing the sequence encoding the carboxy-terminal end
and translation stop followed by BdmHI and EcoRI sites were constructed
resulting in the sequence 5'AGATCTACCCTGTGA
CTCAGCCACTTGCCCCAGTTCATMCCCCATTAGTGTCTMTMGGATCCGMTTC - 3,',
(SEQ ID NO: 3:) where the protein encoding sequence is underlined. The
synthetic fragment was cloned into Bg1II/EcoRI dlgested pS22, resulting in
plasmid pS23. To obtain the recombinant modified B-casein encoding
fragment, three fragments were ligated: an 89-bp PstI/AvdII fragment from
pS24: a 197-bp AvdII/AccI fragment from pS21: and PstI/AccI digested pS23.
The resulting plasmid pS25 was digested with NdeI/BamHI and a 641-bp
fragment was isolated and cloned into the vector pET-3a. The resulting
expression vector was designated pS26.
In order to construct a vector mediating extracellular expression, the
E. co7t signal sequence of the enterotoxin STII gene was introduced in front
of the ,B-casein encoding sequence. A modified STII sequence with NcoI- and
NdeI-compatible ends and an internal C7dI site was obtained by using a
synthetic oligonucleo~ide, 5' -CATGAMMAGAATATCGCAI I I~I I(;I IbCATCGATGTTCGm
mCTATTGCTACMMTGCATATG-3' (SEQ ID NO: 4:). To insert the signal sequence
in front of the ,B-casein encoding sequence, pS25 was digested with
AvdI~EcoRI and a 619-bp fragment was isolated. This fragment was ligated
wi th a syntheti c ol i gonucl eoti de f ragment,
5'CATATGCACGTGAAACCATCGMTCCCTGAGCTCGAG-3' (SEQ ID NO: 5: ), and NdeI/EcoRI-
digested pUC19. The resulting plasmid was designated pS27. The final
expression vector,pS28, was constructed by ligating three fragments: a 700-
bp NdeI/HindIII ,B-casein fragment isolated from pS27, the STII signal
sequence, and a NcoIlHindIII-digested pACAT7 vector.
The expression vectors pS26 and pS28 were used to transform E.co7i
strains BL21(DE3), BL21(DE3)pLysS, and BL21(DE3)pLysE. The bacteria were
grown in Luria Broth medium containing 50 ~g/ml carbenicillin, and when
Bl21(DE3)pLysS and BL21(DE3)pLysE were used the medium was supplemented with
25 /lg/ml chl orampheni col .
For induction of expression the cultures were grown to a density
yielding an optical density (OD) of 0.6 at a wavelength of 600 nanometers
(OD600), then 0.4 mM IPTG was added to induce the T7 system. The cells were
W095/32727 2~ 9~6~ P(l/~).. '.'Q~
harvested about 90 min~tes after inductlon.
Recombinant ~-casein was isolated using standard procedures. The
inducible T7-based expression system resulted in high-level expression of
recombinant ~-casein. Bacteria were harvested and the cells pelletted by
centrifugation. The supernatant contained the periplasmic proteins and the
pellet the cytoplasmic fraction. The recombinant proteins obtained were
compared with native ~-casein, which had been purified by standard methods
including either ion-exchange chromatography followed by reversed-phase HPLC
or gel filtration. Recombinant and native ,~-casein were compared by
standard biochemical techniques comprising SDS-PAGE, Western blotting, amino
acid analysis,peptide mapping, phosphate analysis, and mass spectrometry.
Recombinant ,B-casein expressed in E.co1i was found to comigrate with full-
length, nonphosphorylated native human ~-casein, which is one of seven
native isoforms.
Recombinant human ~-casein has also been expressed in S.cerevisiae
using the pYES 2.0 vector (Invitrogen Corp., San Diego, CA), but the
expression level was approximately 10% of that obtained in E.coli. However,
Hansson et al. found that S. cerevisiae appeared to express phosphorylated
human milk ~-casein.
WO 95/32727
21 9(~609
B-Casel n Hvdrol Ysates
The human B-casein (both native and recomblnant) was digested using
the specific endoproteinase GLU-C (Sigma, sequencing grade) which catalyzes
the hydrolysis of peptide bonds at the C-terminal of glutamic acid residue.
After monitoring the digest using high pressure liquid chromatography, an
enzyme to protein ratio of 1:100 (weight/weight) was chosen for a 30 hour
digestion at 37~C in~O.1 M NH4HCO3, pH 7.8. These digests were dried and
resuspended in appropriate buffers prior to use in the assays discussed
above .
WO95132727 2 1 ~Q9 PCT/I~S9~103628
INHIBITIQN OF HUMAN RSV INFECTION OF HE~-2 ~FI I S
The Long straln of RSV was grown in HEp-2 cells under Eagle minimal
essential medium (MEM) with 2% fetal bovine serum (FBS). The virus was
harvested at a cytopathic effect (CPE) of 3 to 4+, sonicated for 10 seconds
at 50% power with a Microson ultrasonic bell disrupter (Heat Systems -
Ultrasonics, Inc., Farmlngdale, N.Y.), divided into portions and stored at
-70C. The same preparation of virus was used for a series of tests, but
a fresh sample was used for each test run.
The neutralization tests were performed in 96-well, flat-bottomed,
tissue culture, microtiter plates (catalog no. 3596: Costar. Cambridge,
Mass.). Serum or a monoclonal antibody (MAb) in the form of ascites fluid.
which had been heat inactivated at 56C for 30 min., was added to duplicate
wells and serial fourfold dilutions were performed in the microtiter plates.
All dilutions were in MEM-2~ FBS, and the final volume was 75 I~l per well.
Approximately 60 50% tissue culture infective doses of virus in 25 Ill of
MEM-2% FBS then were added to each well. and the mixture was incubated for
2 h at 4C.
Approximately 15,000 HEp-2 cells in 10011l of MEM-5% FBS were added
to each well, and the plates were wrapped in cellophane and incubated at
35.5C in a humidified CO2 incubator for 3 days. The plates were fixed by
aspirating the contents of the wells, washing three times with phosphate-
buffered saline (PBS) at pH 7.2 with 0.5% Tween 20, adding 75 Ill of an 80%
(vol/vol) solution of acetone-PBS, and incubating for 15 min at 4D. After
the incubation period. the contents were aspirated, and the plates were air
dri ed .
The Enzyme Linked Immuno Sorbent Assay (ELISA) was performed on the
same day as the fixation, or the plates were stored overnight at 4C and the
ELISA was performed on the next day. For the ELISA, the wells were
precoated with 200 ~l of PBS with 0.5% gelatin for 30 min at 35C, the
contents were aspirated, the wells were washed with PBS (pH 7.2)-0.5% Tween
20 and 75 ~l o~ bovine anti-RSV serum (BaRSV) Burroughs Wellcome Co.,
Research Triangle Park. N.C.) diluted in PBS 0.5% gelatin plus 0.5% Tween
20 and 2~ normal goat serum was added and incubated for 1 hour at 35.5C.
The contents were aspirated, the wells were washed, and 75 ,ul of peroxidase-
conjugated, goat anti-bovine immunoglobulin G (IgG) (Kirkegaard and Perry
. . , . _, ......
WO 95132727 1/' '7~ 1--
Laboratories. Inc. . Gaithersburg, MD) diluted in PBS-0.5% gelatin-0.5% Tween
20-2% normal goat serum was added and incubated for 1 hour at 35.0C. The
contents of the wells were aspirated again. the wells were washed. and 125
of substrate (0.4 m~ of o-phenylenediamine dihydrochloride per ml 0.015%
H202) in 0.15 M citrate phosphate buffer (pH 5.5) was added and incubated at
room temperature for 40 to 45 min. The reaction was.,stopped with 3.5 M
HCl, and the A490 was read wi th a mi cropl ate reader . Each di l uti on of
antibody was run in duplicate. and each plate included control wells with
uninfected cells. a back titration. i.e. titration of the virus inoculum in
MEM-2% FBS. and a titration of a preimmune or nonneu~ralizing antibody. An
absorbance reading of greater than or equal to 3 standard deviations above
the mean of 15 control wells~was considered to be evidence of virus
replication. The dilutions of BaRSV (1:1.000) and goat anti-bovine IgG
(1:5,000) used through the study initially was determined by checkerboard
titrations .
This assay was based upon the disclosure of Anderson et al..
"Microneutralization Test for Respiratory Syncytial Virus Based on an Enzyme
Immunoassay". JOURNAL OF CLINICAL MICROBIOLOGY, December 1985, pages 1050-
1052.
WO 95/32727 2 ~ 9 ~ 6 ~ 9 PCTIUS95103628
.
RESULTS FROM HEp-2 CELL ASSAY
The human and bovine,B-caseln solutions were prepared first in 20 mM
ethanol ami ne . 6 M urea, pH 9 . 5 and then washed twi ce i n PBS by
ultrafiltration using Centricon membrane filters (Amicon. MA) with a cut-off
of 3,000 daltons. After resuspending in appropriate buffer for the HEp-2
cell assay described above. these samples were tested in the assay.
Experiments with different designated numbers were performed in different
days. As shown in Table 1, human ~-casein caused an inhibition of
infection/virus replication of 50% or more at concentrations of 0.4 mg/ml
or greater. It should be noted than when referring to Table 1, a higher
percent inhibition indicates a higher level of bioactivity of the "AGENT".
and a lower percent inhibition indicates a lower level of activity of the
"AGENT". Bovine ~-casein was not sign~ficantly active even at 1.6 mg/ml.
These results indicated that ~-casein from human milk has different
bioactivity compared to the bovine milk ~-casein.
WO 95/32727
21 9060q
TA8LE 1
INHIBITIQN OF HUMAN RSV INFECTION OF HEp-2 CELLS
AGENT USED CONC. PERCENT INHIBITION
(mg/ml )
~-Casein Isolated 1.6 >90
from Human Milk
0 . 8 >90
0.4 >90
Bovine ~-casein 1.6 0
0.8 0
0.4 0
Data shown are average of three replicates.
WO95/32727 2~ 9~9 r~".l~ /Q~
INHIBITION OF HUMAN RSV INFECTION OF LLC-MK2 r~F~
The RSV inhibition assay quantitatlvely determines the ability of a
test reagent (antibody or other bioactive compound) to inhibit the
infection of monkey kidney cells (LLC-MK2) (ATCC CCL 7) in microtiter
plates. Infected cells were identified using an immunoperox1dase method.
The method is described briefly below.
LLC-MK2 cells were seeded into fibronectin treated Costar microtiter
plates (5.0 X 103 cells per well) and incubated for 3-4 days prior to use
in the infectivity reduction assay. On the day of assay the Long stra1n
of RSV was diluted in MEM to 10-20.000 infected cell units (ICU/mL). and
added to an equal volume (200,uL) of serially diluted sample preparations
at suitable concentration (ex. 0.5, 1.0, and 2.0 mg casein/mL). Mixtures
of diluted test samples and virus were then incubated for 2 hours at 4ac
prior to adding to LLC-MK2 cells. Prior to addition of the diluted sample-
virus mixtures to microtiter plates. culture medium was removed and the
monolayers rinsed one time with MEM. All diluted sample-virus mixtures were
tested in tr1pl1cate wells. The diluted sample-virus mixtures were allowed
to absorb to LLC-MK2 monolayers for 2 hours at 37C in a humid1fied CO2
incubator. Following incubation. 150 ~l of MEM was added to all wells and
the pl ates i ncubated at 37C for 16 hours i n the COz i ncubator . After
overnight incubation. the culture medium was removed and the monolayers
fixed with cold ethanol. After fixing. microtiter plates were rinsed once
with 200 1~1 Dulbecco s PBS per well. and bovine anti-RSV antibody (200 ~l)
was added to all wells. Following a 30 minute incubation at room
temperature and three rinses wlth PBS/0.5% chick albumen (PBS/CEA)
peroxidase labeled rabbit anti-bovine IgG was added to all wells and
incubated at room temperature for 30 minutes. Microtiter plates were then
rinsed 3 times with PBS/CEA and diaminobenzadine substrate added and
incubated for 20 minutes. Plates were then rinsed as above with PBS/CEA.
and the number of stained RSV-infected cells per well determined using an
i nverted mi croscope.
WO 95/32727 2 1 9 0 6 ~ 9 PCTIUS95103628
12
RESULTS FROM LLC-MK2 CELL ASSAY
The proteins described in Table l were also tested in this assay for
inhibition of RSV infection. Results from this assay are shown in Table 2.
Once again. native human milk ,B-casein was found to be active at
concentrations of 1 mg/ml or greater while bovine~ ,B-casein was not
significantly active. The GLU-C hydrolysates of both native and recombitant
human ~-casein were active at concentrations of 0.75 mg/ml and higher.
Hence these results indicated that the recombinant human ,~-casein native
human ,B-casein and their hydrolysates inhibit RSV infectlon of both HEp-2
mammalian cells and LLC=MK2 mammalian cells.
W095132727 2 i ~6~q r~l,u~ 6~
TABLE 2
INHIBITION OF HUMAN RSV INFECTION OF LLC-MK2 CELLS . -.
AGENT U~ED CONC. PERCENT INHIBITION
(mg/ml )
-Casein Isolated from 1.5 87
Human Mi l k
69
0 . 75 33
O . 38 20
Bovi ne ,~-casein 1 21
0.5 23
0.25 6
Hydrolysate of ~-casein 1.5 99
Isola~ed from Human Milk
0 . 75 77
O . 38 60
Hydrolysate of Recombinant
Human,B-Casein 1.5 84
0 . 75 42
O . 38 25
GLU-C Enzyme Control .025 64
. 0125 27
. 0068 19
Data shown are average of four replicates.
w09~132727 2 l 9 0 6 0 9 P._l/I.lJ, ~/Q~f.7f
It has been concluded from the foregping experiments that ~-casein
isolated from human milk, a recombinant form of the ~-casein contained in
human milk, and hydrolysates of both. inhibits the infection of mammalian
cells by RSV. Furthermore, inasmuch as RSV has been identified in the
literature as being associated with respiratory tract infection, it has been
concluded that the above identified forms of human ,B-casein may be employed
in the preventlon and treatment of respiratory tract infection in humans,
especially in human infants. In view of the therapeutic effect of enterally
ingested human milk cQntaining human ,B-casein upon respiratory tract
infection, it is concluded that the above identified forms of human ,B-casein
have a therapeutic benefit when enterally (orally) ingested.
The therapeutic effects described in the preceding paragraph may be
provided by an enteral liquid nutritional product. such as infant formula,
comprising one or more proteins not contained in human milk in combination
with a therapeutically effective amount of at least one of the forms of
human ,~-casein described in the preceding paragraph. It is further
concluded that the infection of mammalian cells by RSV may be inhibited by
administering via a nasal passageway, or as a throat spray, a formulation
containing a therapeutically effective amount of at least one of the forms
of human ,B-casein identified in the preceding paragraph. Such a nasally
administered formulation may be in the form of either drops or a spray.
The enteral nutritional, throat spray and nasal products and methods
are believed to be effective in inhibiting the infection of mammalian cells
by RSV because the interaction of the human ~-casein and RSV is believed to
occur via direct contact rather than following digestion and absorption of
the ~-casein.
It is believed that the above identified forms of human ~-casein may
be i ncorporated i nto any standard or speci al i zed enteral 1 i qui d nutri ti onal
product containing at least one protein not found in human milk, such as
bovine milk based or soy based infant formulas, and other beverages consumed
by young children. In a preferred embodiment no proteins or hydrolysates
thereof found in human milk, other than ~-casein, are contained in the
liquid enteral nutritional product. Such a product has utility in the
treatment and preventipn of respiratory tract infection in human infants.
While preferred embodiments of the invention have been disclosed~ it
will be apparent to those skilled in the art that various changes and
WO 95/32727 2 ~ 9 ~ 6 ~J q r~ ;?12
modiflcations may be made therein w~thout deviating from the spirit or scope
of this invention.
WO 95/32727 2 ~ 9 1) 6 0 9 P~ 1 / u~ ~ ~7~ ~
16
SEQUENCE LIST[NG
( I ) GENERAL INFORMATION:
(i) APPLICANT: Abbùtt T.~hl~ratnri~c
(ii) TITLE OF ~NVENTION: Inhibition of Infection of Marnm~lian Cells b
Respiratory Syncytial Vilus
(ii~i) NUMBER OF SEQUENCES: 5
(i~) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LonnieR Drayer
ROSS Products Division
Abbott I .~hnr~toripc
(B) STREET: 625 Cleveland Avenue
(C) CITY: Colurnbus
(D) STATE: Obio
(E) COUNTRY: United States of America
(F) ZIP: 43215
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 Mb storage
(B) COMPUTER: IBM Cornpaùble
(C) OPERATING SYSTEM: MS-DOS Version 6.21
(D) SOFTWARE: WordPerfect Version 6.0a
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FTLING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
Wo 9s/32727 ~ 1 9 () 6 0 9 P~
(A) APPLICATION NUMBER: US 08/249,554
(B) FILlNG DATE: 26-MAY-1994
(A) APPLICATION NUMBER: US 08/249,555 - --
(B) FILINGDATE: 26-MAY-1994
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (614) 624-3774
(B) TELEFAX: (614) 624-3074
(C) TELEX: None
(2) INFORMATION FOR SEQ rD NO: I
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1065 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: SDIgle
(D) TOPOLOGY: UDkDowD
(ii) MOLECULE TYPE: Cloned cDNA ~ , the product of a
humaD geDor~ic DNA segmeDt
(A) DESCRIPTION: HumaD miLk ~eta-case
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE: ~~
(vi) ORIGINAL SOURCE: Human
(A) ORGANISM: Homo sapieDs
(B) STRAIN:
(C) rNDlVIDUAL ISOLATE:
wogs/32727 2 ~ 90609
18
(D) DEVELOPMENTAL STAGE: Adult
(E) HAPLOTYPE:
(F) TISSUE TYPE: Man~mary gland
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE: Human Mammary Gland
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAMElKEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis
(D) OTHER INFORMATION: The encoded product of nucleotide SEQ ID
NO: I is the hunlan milk protein, ,~-casenn.
(x) PUBUBLICATION INFORMATION:
(A) AUTHORS: B. LonnerdaL et al.
(B) TITLE: Cloning and sequencing of a cDNA encoding human milk beta-
casein.
(C) JOURNAL: Federation European P ~ I ' SocietyLetters
(D) VOLUME: 26g
wo g5132727 2 1 q 0 6 0 9 PcTIUssS103628
19
(E) ISSUE:
(F) PAGES: 153-156
(G) DATE: 1990
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: I
CGG ATG AAG GTC CTC ATC CTC GCC TGC CTG GTG GCT CTT GCT CTT - 45
GCA AGG GAG ACC ATA GAA AGC CTT TCA AGC AGT GAG GAA TCT ATT ~ 0
ACA GAA TAC AAG AAA GTT GAG AAG GTT AAA CAT GAG GAC CAG CAG 135
CAA GGA GAG GAT GAA CAC CAG GAT AAA ATC TAC CCC TCT TTC CAG 180
CCA CAG CCT CTG ATC TAT CCA TTC GTT GAA CCT ATC CCC TAT GGT 225
TTT CTT CCA CAA AAC ATT CTG CCT CTT GCT CAG CCT GCT GTG GTG 2 7 0
CTG CCT GTC CCT CAG CCT GAA ATA ATG GAA GTC CCT AAA GCT AaA 315
GAC ACT GTC TAC ACT AaG GGC AGA GTG ATG CCT GTC CTT A~A TCT 3 6 0
CCA ACG ATA CCC TTT TTT GAC CCT CAA ATC CCA AAA CTC ACT GAT 405
CTT GAA AAT CTG CAT CTT CCT CTG CCT CTG CTC CAG CCC TTG ATG 450
CAG CAG GTC CCT CAG CCT ATT CCT CAG ACT CTT GCA CTT CCC CCT . 4 9 5
CAG CCC CTG TGG TCT GTT CCT CAG CCC AAA GTC CTG CCT ATC CCC 540
CAG C~A GTG GTG CCC TAC CCT CAG AGA GCT GTG CCT GTT CaA GCC 5 Z 5
CTT CTG CTC AAC CaA GAA CTT CTA CTT AAC CCC ACC CAC CAG ATC 63 0
TAC CCT GTG ACT CAG CCA CTT GCC CCA GTT CAT AAC CCC ATT AGT 675
GTC TAA GAA GAT TTC A~A GTT AAT TTT CCC TCC TTA TTT TTG AAT 720
TGA CTG AGA CTG GAA ATA TGA TGC CTT TTC CGT CTT TGT ATC ACG 765
TTA CCC CAA ATT AAG TAT GTT TGA ATG AGT TTA TAT GGA AAA AAT 810
GAA CTT TGT CCC TTT ATT TAT TTT ATA TAT TAT GTC ATT CAT TTA ~55
ATT TGA AAT TTG ACT CAT GAA CTA TTT ACA TTT TCC AAA TCT TAA 9 0 0
TTC AAC TAG TAC CAC AGA AGT TCA ATA CTC ATT TGG AAA TGC TAC 945
A~A CAT ATC AAA CAT ATG TAT ACA AAT TGT TTC TGG AAT TGT GCT 9 9 0
TAT TTT TAT TTC TTT AAG AAT CTA TTT CCT TTC CAG TCA TTT CAA 10 3 5
TAA ATT ATT CTT AAG CAT AAA AAA AAA A~A 1065
(2) INFORMATION FOR SEQ ID NO: 2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 base pairs
Wo9S/32727 2 ~ 906 0q P~ 7~ ~
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Unkuown
(ii) MOLECULE TYPE: Synthetic 'i,,
(A) DESCRIPTION:
(iii) HYPOTHETICAL
~ ) ANTI-SENSE:
(~) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: Synthetic O'i~ Sequence
(A) ORGANISM:
(B) STRAIN:
(C) rNDlVlDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANEILE:
(vii) IMMEDL9TE SOURCE: _
(A) LIBRARY:
(B) CLONE:
(vui) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
wo gsl32727 2 ~ ~ ~ 6 0 9 1~ L L9.
(C) UNlTS:
(rx) FEATURE:
(A) NAMEIKEY:
(B) LOCATION:
(C) lDENTrFlCATlON METHOD: DNA sequencing and restriction analysis
(D) OTHER INFORMATION: The synthetic n~j~ ^IP~ assures adaptation of
translation sequence to E. coh codon usage.
(x) PUBUBLICATION rNFORMATlON:
(A) AUTHORS: L. Hansson, et al.
(B) TITLE: Expression of Human Milk ~-casein in Escherichia coli: Comparison of
Rf - ' Protern witb Natrve Isoforms.
(C) JOURNAL: Protern Expression and Purification
(D) VOLUME: 4
(E) rssuE:
(Fl PAGES: 373 - 381
(G) DATr~`: 1993
(H) DOCUMENT NUMBER:
(I) FlLrNG DATE:
(J) PUBLICATION DATE:
(K) Rr LEVANT RESrDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2
CTG CAG AAT TCA TAT GCG TGA AAC CAT CGA ATC CCT GAG CTC GAG 4
CGA AGA ATC GAT CAC CGA ATA CA~ A~A AGT TGA A~A AGT TAA ACA ~ O
CGA GGA CCA GGA TCC 10
(2) rNFORMATlON FOR SEQ rD NO: 3
(i) SEQUENCE CHAr~ACTERlSTrCS:
Wo 95/32727 2 1 9 0 6 ~ ~ r~l,o~
(A) LENGTH: 71 base pairs
(B) TYPE: Nucleicacid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Unknown
(ii) MOLECULE TYPE: S~ntbetic rli, ' '-
(A) DESCRIPTION:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGlNAL SOURCE: Syntbetic O~ '~ Sequence
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(~) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBR~RY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
2 1 9060q
Wo 9~/32727 r~ Jsg~lu~628
23
(B) MAP POSmON:
(C) UNITS:
(rx) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) rDENTlFICATlON METHOD: DNA sequencing and restriction analysis
(D) OTHER INFORMATION: The synthetic ~ encodes the carbox~-
terminal end and tranlation Stop.
(x) PUBUBLICATION INFORMATION:
(A) AUTHORS: L. Hansson, et al.
(B) TITLE: Expression of Human Milk ~-casenn in Escherichia coh: Comparison of
Protenn with Native Isoforms.
(C) JOURNAL: Protein Expression and Purification
(D) VOLUME: 4
(E) ISSUE:
(F) PAGES: 373 - 381
(G) DATE: 1993
(H) DOCUMENT NUI\~BER:
(I) FILTNG DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQIJENCE DESCRIPTION: SEQ ID NO: 3
AGA TCT ACC CTG TGA CTC AGC CAC TTG CCC CAG TTC ATA ACC CCA 45 - =~
TTA GTG TCT AAT AAG GAT CCG AAT TC 71
(2) INFORMATION FOR SEQ ID NO: 4
_ . . . . . . .
wogs/32727 2190609 r~llu~ fi7~ ~
24
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 68 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Unlmown
(ii) MOLECULE TYPE: Synthetic
(A) DESCRIPTION:
(iii) HYPOTHETICAL:
ANTI-SENSE:
(v) FRAGMENT TYPE,
(vi) ORIGINAL SOURCE: Syn~etic O'i,, ' '- Sequence
(A) ORGANISM:
(B) STRAIN:
(C) INDIV~DUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(~I) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
wo 95/32727 2 ~ 9 1) 6 0 9 ~ s~ x
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis
(D) OTHER INFORMATION: Modified enterotoxin STII signal sequence.
(x) PUBUBLICATION INFORMATION:
(A) AUTHORS: L Hansson, et al.
(B) TITLE: Ex,oression of Human MiLk ~-casein in F~r~Tirhi~ coli: Co~arison of
Protein with Native Isofor~ns.
(C) JOURNAL: Protein Expression and Purification
(D) VOLUME: 4
(E) ISSUE:
(F) PAGES: 373 - 3x 1
(G) DATE: 1993
(H) DOCUMENI NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ rD NO: 4
CAT GAA AAA GAA TAT CGC ATT TCT TCT TGC ATC GAT GTT CGT TTT 45
TTC TAT TGC TAC A~A TGC ATA TG 68
(2) INFORMATION FOR SEQ ID NO: 5
WO9~/32727 2 ~ 9 0609 r~ q~
26
(i) SEQUENCE C~iARACTERlSTlCS:
(A) LENGTH: 37 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Singie
(D) TOPOLOGY~ Unknown
(ii) MOLECULE TYPE Synthetic
(A) DESCRIPTION:
(iii) HYPOTHETICAL:
(i~) ANTI-SENSE: :
(v) FRAGMENT TYPE:
(vi) ORiGlNAL SOURCE: Synthetic O'i" I ' Sequence
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LrNE:
(I) ORGANELLE:
(vii) IMMEDiATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
~ woss/32727 21 q0609 r~l,o~s~
(A) CHROMOSOMEISEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE: =
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD: DNA seqnencing and restriction analysis
(D) OTHER INFORMATION:
(x) PUBUBLICATION INFORMATION:
(A) AUTHORS: L. Hansson, et al.
(B) TITLE E~ression of Human Milk ~-casein nn Escherichia coli: Cornparison of
R~ ' Protein with Native Isofornns.
(C) JOURNAL: Protein Expression and Purification
(D) VOLUME: 4
(E) ISSUE:
(F) PAGES: 373 - 381
(G) DATE: 1993
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:
CAT ATG CAC GTG A~A CCA TCG AAT CCC TGA GCT CGA G 3 7 - -~
