CA2068533A1 - Control of secretion of milk - Google Patents

Abstract

A protein which inhibits milk secretion by lactating cows and which is present in the second (2A) significant peak when a nominally 10-30 KDa fraction of the whey proteins of the milk is resolved on a ''Mono Q'' anion exchange column using 10 mM imidazole buffer, pH 7.0 and a sodium chloride elution gradient.

Description

2Q~8~33 . `,' ;

CONTROL OF SECRETION OF MILK
Backqround of the invention ...
1. Field of the invention . ~.`
This invention relates to a newly isolated protein from cow s .
milk and the use of the protein or antibodies thereto for the 05 control of milk secretion in lactating animals.

2. Qescr~tion of the Qrior art The rate of milk secretion by a lactating animal is regulated by the frequency of milk removal. In other words there is a mechanism which acts to match the animal s supplv of milk to the demand of her offspring or of a farmer s milking regime. Part of this control is achieved by the. release of galactopoietic :
hormones during suckling or milking. However studies by workers .
at the Hannah Research Institute Ayr Scotland on lactating goats have shown that another factor is involved. ~his ~s an inh~bitor wh~ch decreases milk secret~on at a local level i.e.
at the individual gland of an udder.
It has already been shown that the ~nhib~tor is present in a goat milk fraction containing whey prote~ns of molecular weight 10-30 KDa th~s range of molecular we~ghts being determined by 20 the nomlnal slzes of f7.1ters used ln ultraf~ltratlon of the ~ .
whey. The effect has been demonstrated both in ~ Q and in vivo. The in v7tro technique described by C.J. Wilde et al.
B7Ochem. J. 242 285-288 (1987) consists in culturing explanted pieces of rabbit mammary with and without the milk fraction and .-25 demonstrating the inhibition of lactose and casein synthesis. .:
See also G.M. Stewart gt a7.. J. Endocrinology 118 Rl-R3 (1988). In the in v o technique. C.J. Wilde et al. Ouarterlv Journal cf Experimental Physiolog~ 73. 391-397 (1988) the mi lk fraction was iniected into a sinc7e mammarv gland of aoats via ~ 30 the teat canal. A temporarv dose-dependent reduction of mil~ ;
; -~ vield specific to that gland was observed.
It has remained a problem to determine whether an inhibitor -is present in cow s milk and if so to purify it sufficiently for identification with a view to chemical or biological ~ 35 svntl)esis.

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W o 91/07439 PCT/GB90/01743 . .

, Summarv of the invention It has now been found that lnhibitory activity is present in a 10-30 KDa fraction of whey from cow's milk. that this fraction can be separated by anion exchange FPLC (Fast Protein Liquid 05 Chromatography) into a number of peaks and that the inhibitory activity is concentrated mainly in two particular peaks out of eight.
There are various ways of defining the proteln of the 1nvention, of varying degrees of reliab~l~ty. One currently preferred definition is a protein which inhibits milk secretion by lactatina cows and which is present in the second significant peak (labelled "2A" in Figure 1~ when a nominally 10-30 KDa fraction of the whey proteins of the milk is resolved on an anion exchange column containing particles of monodisperse hydrophilic polymers having pendent -CH2N(CH3)3+ groups, the particle diameter being 10 ~ O.S ~m, esPecially a "Mono Q" column, using ~0 mM imida201e buffer, pH 7.0 and a sodium chloride elution gradient.
The molecular weight of the pea~ 2A proteln as determlned by gel filtration chromatography is about 7 KDa. When the peak 2A
protein ls further resolved, by gel flltration chromatography on a cross-l~nked agarose gel havtn~ a partlcle s~z~ of 10~2 ~m, such as "Superosei', the ma~or component, wh~ch is that- which ~ppears second, comprises the inhibitory protein. Further when peak 2A (or its equivalent in wh.ich the whey is not fractionated before anion exchange chromatoaraphy~ is resolved on a chromatofocussina column containin? Darticles of monodisperse hydrophilic polymers having pendant tertiary (-N+HR2) - and quaternarv (-N+R~) amine groups where R represents an organic group, the particle diameter beina lo ~ 0.5 ~m espec~ally a ~; "Mono "P column, usina lQ mM imidazole. pH 6.5 and amphoteric ~: buffer of pH 4.0 to create a pH aradient of 6.5-4.0, the protein ~: is present in the second significant peak ("2A.2").
Any combination of one or more of the above features, together with the inhlbitorv action of the protein, might be .. .,. . .,. .. ., .,.. . .. , ... . . - - ;.-- - ~ .,- -. .. , .- -. - . . . . - . . .

W O 91/07439 PCT/GB90/01743 ~ ~
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sufficient to define the protein uniquely and accordingly .
applicant does not wish to be limited unnecessarily to specific combinations, in case one of them or some aspect of one of them miaht later be re-determined and found not sufficiently to 05 approximate to their definition given above, while the remaining .
features are confirmed, and leave no doubt as to the identity of :
the protein Precisely which features are the most meaningful and the most rellable are, in anv case, a matter of judgement, the preferred deflnitlons given above reflect~ng appl~cant s current judgement. It will be aDpreciated, therefore, that the protein defined by other combinations of features herein set forth is to be considered as encom~assed by the invention. .:
.
A property which might be useful for defining the protein is .. : .
the isoelectric point (pI). It has been found that peak 2A gives .
a pI in the range 4.8 to 5.0 ~hen determined in a tube of po1yacrylamide gel, while peak 2A.2 gives a pI of 4.9-5.0 determined by isoelectric focussing.
The prote~n can be in glycosylated or unglycosylated form. ;
Ant~bod1es to the prote~n, whether polyclonal, monoclonal or englneered, are w~thin the scope of this invention.
Where nat~onal patent law permits, the administration of the inhlbltor to decrease mllk ~eld or an ant~body thereto to suppress at least partly the actlon of the lnh~bitor, to cows or other animals is within the invention.
3rief descriDtion of the drawinqs Figure l shows the resolution of the 10-30 KDa fraction by anion-exchanae chromatography: ~ -Figure 2 and 3 show the ~urther resolution of two of the peaks of the Fiaure l anion evchanae chromatoaraphv by gel 30 filtration chromatoaraphv: and -.
Fiaure 4 shows the further r^;olu,ion of one cf the peaks obtained from anion-exchanae chromatoaraDhy bv chromatofocussing.
DescriDtion of the Dreferred embodiments The protein of the invention e~ists in cow s milk, probably in glvcosvlated form. It is believed that the effect of .

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2~68~33 glycosylation is simply for attachment of the protein to the appropriate cells within the mammary gland. It would be expected, therefore, that the protein could be administered locally to the gland in an unglycosylated form. .
05 In relation to goat s milk, using a 10-30 KDa fraction of whey protein, it has been demonstrated that the inhibition of lactose and casein synthesis in mammary explant culture is dependent on the dose of the ~nhibltor-contaln~ng fraction.
~urther, when the explants have been exposed to the lnhibitor-containing fraction, washed and re-cultured ln fresh medium in the absence of the inhibitor, the capacity to synthesise lactose and casein is recovered. In vivo, it is found that administration of the protein to the mammar,v gland causes the milk yield to decrease within hours, with full recovery of yield 24-36h after a single administration. However, when a change in milking frequency - and therefore autocrine control -was sustained over weeks, there was an effect on the synthetic capacity i.e. degree of differenttat~on of the secretory cells attr~butable to the autocrine inhibitor. These long-term effects on mammary cell activ1ty are accompanied by changes in the number Of cell-surface hormone receptors for prolact~n. Thrice~dally mllkina of lactating goats for 4 weeks ~ncreases Sell actlvity and prolactin receptor number per cell, whereas a decrease in mllking efficiency extending over 21 weeks reduces secretory cell differentiation and prolactin receptor number. Therefore, these long-term effects, and also the acute regulation bv the autocrine - :
inhjbitor of the invention could be due primarilv to modulation of the sensitivity of individual alands to endocrine control.
There is everv reason to believe that the same effects will be demonstrable in relation to the protein of the invention obtained from co~ s milk, in relation to cows.
Antibodies can be raised against the protein of the invention by any conventional methods, e.g. as polyclonal antisera, mouse -.
monoclonal antibodies, cow-mouse hvbrid monoclonal antibodies or ~ :.
as engineered antibodies, bv anv of the currentlv available w o 91/07439 PCT/GB90/01743 -, 2068~33 ,,~

methods. Passive immunisation methods can then be used to generate a reduction in the effect of the natural inhibitor. when :
this is desired in order to increase milk yield. Frequently however there will be a need to reduce milk yield in order to 05 meet milk quotas in which event the inhibitor itself is administered. Conventional carriers and adjuvants known in -. ; -vaccination can be used.
The inventlon is applicable to any animal respons~ve to the ~nh~bltor defined here~n. Slnce the 10-30 KDa goat s milk fraction has been successfully found to reduce milk accumulation and relevant enzyme activities when injected into the mammary gland of rabbits it is likely that the cow s milk inhibitor will be effective in some other lactating animals.
For intraductal injection into the mammary gland a dose in the range of l to 50~9 especially 5 to 20~9 of inhibitor is l~kely to be effective and should be repeated as required e.g.
daily and possibly reduced when given over long periods.
The protein of the invention can be obta~ned from cow s milk by the method descr~bed in the Example or some var~ant thereon.
It can be recovered in pure form from an eluate by extensive d~alysls aga~nst water ~uslng an appropr~ate membrane for ~;
retentlon of the protein e.g. wlth a nomtnal molecular welght cut-off of about 6 KDa) and freeze-drying. However it is expected that it would be synthesised by protein synthesis or by a recombinant DNA method.
The following Example illustrates the invention.
EXAMPLE
This Example describes the preDaration and properties of the inhibitor of the invention.
l. PreDaration of cow milk fractioni Milk was obtained at the morning milking from Friesian cows~
and was defatted by centrifugation 500 9 15C 20 min~ and filtered through glass wool. Defatted milk was centrifuged (80 000 9 15C 2h) yielding a pellet of casein micelles and a clear supernatant containing whev proteins. The whey fraction `:

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20~8533 6 -..
was dialysed against distilled water for Z4 h. -The whey fraction was subjected to ultrafiltration using a .:
filter with a nominal cut-off value of molecular weight 30,000 :~
Daltons (Da). The filtrate obtained with the 30,000 Da filter , .
05 was concentrated by ultrafiltration with a 10,000 Da filter. The - .
10,000-30,000 Da fraction was dialysed extensively against water, sterilized by filter sterilization and concentrated by freeze-dry~ng for anibn exchange chromatography.
2. An~on exchanqe chromatoaraphv of cow _b~y_~Q~eins The 10-30 KDa whey fraction was resolved on a MonoQ HR
10/10 anion exchange column ~Pharmacia) uslng an FPLC
chromatography system (Pharmacia). The whey fraction was dissolved in 10 mM imidazole at four times its concentration in the original milk and the pH adjusted to 7Ø Before chromatography, the sample and buffer (degassed) were filtered through 0.2 ~m filters. 1 ml of the 4 x concentrated whey fraction was loaded for each separation; the flow rate was 4.0 ml/min. A sodium chlorlde elution gradient was used.
Fract~ons containing proteln peaks eluted from the column .
20 were d~alysed extensively against distilled water, freeze-dried :
and stored at -20C, before use in the next stage.
Figure 1 of the drawings shows the elutlon of protein from the chromatography column. Protein concentrat~on, as absorption of light at 280 nm, on the left-hand ordinate is plotted against cumulative volume of eluted material on the abscissa. The plot is shown as lines. The right-hand ordinate is calibrated to show the sodium chloride gradient, from n to l.OM. used in the eluant. The broken line is a plot of the sodium chloride concentration. The peaks are iabelled Vo = void volume containing material not bound by the column and then in order of elution. the peaks being numbered a,; fa.r as possible by a system . .
which relates them to those obtained when the non-ultrafiltered -whey protein is chromatographed bv the same method. The . .
numbering is 1, 2A, 2B 3, 4, 5, 6A, 6B. 6C, 7 and 8 (although the last two peaks do not correlate with those for the ' .. , . , . , ", ~ . .. , .. ., , . ., , , , , . - - . ~ . . - , - . . - , W O 91/07439 PCT/GB90/01743 ~ -20~8533 _ 7 -non-ultrafiltered whey).
3. Mammarv ex~lant bioassav of milk fractions :
Mammary tissue was cultured as explants small pieces of -parenchymal tissue approximately lcm3 and weighing 0.5-0.7 mg.
05 Explants were prepared from mammary tissue of mid-pregnant New Zealand White rabbits as described by R. Dils & I.A. Forsyth in Methods ~n Enzymology 72 724-742 (1981). The explants were cultured in a defined culture medlum ~Medlum 199; G~bco Europe Ltd. Palsley UK) on stainless steel grids each holding 30 explants so that the explants were in contact wlth the medium but not completely submerged in it. The medium was supplemented throughout with insulin (5~g/ml). cortisol (lOOng/ml) and -prolactin (l~g/ml). Explants were cultured in this medium under an atmosphere of air/C02 (19:1 v/v) for 42 h with replenishment of medium after 24 h. At thls time groups of exp1ants (3 or 4 groups per treatment) were transferred into fresh medium containing hormones and one of the fract~ons of cow milk under test. The milk fract~ons tested ~n th~s exper~ment were obtained from cow s m~lk whey wh~ch had not been ultraf~ltered ~as dlst~nct from the 10-30 KDa fract~on referred to above) but whlch had been fract~onated by anion exchange chromatography as described above. They were d~ssolved ~n 10 mM He~es pH 7.4 at tw~ce their concentrat~on in the original m~lk and added to an equal volume of two times concentrated culture medium so as to ~5 be at 100% of their oriainal milk concentration in normal strength culture medium. Control cultures. containing only the diluent for the mil~ fractions. were included in each experiment. Average -ates of lactose and casein synthesis during a furtle~ culture in the presencD or absence of mil~ fraction . -were measured bv tle addition o~ tU-14C]alucose (U=uniformlv labelled: ~.1 mCi/mmol) and b-[4. -3H]leucine (........ 2mCi/mmol) :~ -respectivelv in this culture medium. At the end of the 6 1 period~ explants and culture medium were separated and stored frozen in liquid nitrogen.
Explants were homoaenized at 4C in 1.0 ml of lOmM ~ris/ HCl ` ' . . . ' ' '. ' ' . , ' ~' " ".' - . ,~ . ' . ' ' .
'' , ' . "' ' " , ' ' ' .. :, - ,', . '' . ~' ' ' ' : ; ' . ' w O 91/07439 PCT/GB90/01743 2068~3~

pH 7.0, containing 5mM ethylenealycol-bis-(2-aminoethyl ether N,N,N ,N -tetraacetic acid (EGTA) and 2mM phenylmethane-sulphonyl fluoride by 10 strokes with a glass/PTFE homogenizer, followed by sonication for 30s (Kontes ultrasonic cell disruptor, .
05 30% maximum power), and a particle-free supernatant was prepared by centrifugation at lO,OOOg for 5 min. 3H-labelled casein was prepared from the particle-free supernatant by precipitation at ~ts isoelectr~c point, and the prec~pitate was subjected to SDS-polyacrylamide gel electrophoresis, as described by 10 C.J. Wilde et al., Exp. Cell Res. 15~, 519-532 (1984). 8ands corresponding to casein polypeptides were visualized by staining with Coomassie brilliant blue, and were excised and counted for [3H] radioactivity as described by S.M. Russell et al., Biochim.
Biophys. Acta 714, 34-45 (1982). ['4C] lactose was selectively precipitated from explant homogenates and culture medium using ethanol/d~ethyl ether (3:1, v/v), N.J. ~uhn & A. White, Biochem.
J 1~. 77-84 (197S) and the radioactivity of the precipitate counted. Results were corrected for carry-through of ~14C~
glucose from culture medium ~usually < 0.08X), by measuring t14C]
rad~oactivity after extract~on of uncultured medium. The addltion of milk fract~ons d~d not affect the dlstr~bution of secreted products between the extracellular space of the explants and the med~um.
The amount of radioactive material (casein and lactose) was 25 expressed as a percentage of that produced by the explants to ~ -which no milk fraction had been added. The results are shown in Table 1. The figures in parenthesis are the numbers of experiments performed on various peaks.

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W o 91/07439 PCT/GB90/01743 : -' 20g8 5:~3 ~.' . ' ' _ 9 _ ., .
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Peak Lactose synthesisCasein synthesis ;
number (% of control)(7O of control) . . .
No addition (control) 100 100 1 96.8 + 3.7 (5)90.6 + 3.5 ~5) 2(~2A ~ 2B~65.8 ~ 7.5 (6)~63.1 1 14.2 (6)*
3 94.6 ~ 4.6 (S)86.7 ~ 12.2 (S) 4 104.0 ~ 6.0 (5)146.4 + 22.8 (5) s 102.1 + 1.7 (4)94.3 + 21.3 (5) 7 85.3 + g.8 (4)91.6 + lg.8 ~5) 8 90.4 ~ 4.8 (3)94.4 + 6.1 (5) 9 110.9 ~ 5.6 (3)90.3 + 10.8 (4) 102.0 (2) 120.3 (2) Test vs control; p<0.05 ~paired t-test) From the Table it will be seen that peak 2 was the more actlve than the others ln inhibiting lactose synthesis, a major determlnant of milk yield, and casein synthesis.
The above exper~ments were then repeated, using fract~ons OS from peaks 2A and 2B, wlth the results shown ~n Table 2 below.
, .
TABLE Z
Peak ~ Lactose synthesisCasein synthesis ~-number (% of controls)(% of controls) No addition .
~ (contol) loo 100 s~ 2A 71.4 + .1 53.9 _ 5.9 2B 80.9 + 9.6 80.8 + 6.6 Res,lts are the mearl + s.e.m. for 3 experiments.

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, . , o It will be seen that both fractions were inhibitory to some extent although lack of clear separation of the inhibitor component between peaks 2A and 2B accounts for some of these effects.
05 4. Gel filtration chromatoqraDhv of Deaks Gel filtration of each of peaks 2A and 2B prepared from the 10-30 KDa fraction as described above was carried out using an FPLC chromatography system and a Superose 12 HR 10/30 column (Pharmac~a). Superose 12 ~s a h~ghly cross-linked agarose matrix with a particle size of 10+2 ~m and an exclusion limit of 2 x lo6 Da. The buffer was 50 mM Tris/HCl pH 7.5 containing 100 mM KCl which was filtered (0.2 um filter) and degassed before use. Samples (routinely 1-10 yg in a maximum volume of 200 ~
were dissolved In the same buffer and filtered before use ~0.2 ~m filter). The column was calibrated using molecular weight standards in the m.w. range 200 000-12 400 (Sigma MW-GF-200 kit) and also aprotin~n ~molecular weight 6 500) .and bovine . .
a-lactalbumin ~molecular weight 14 200). Calibration curves of logtmolecular welght] versus Ve/VO were prepared where VO ~ void volume and Ve elut~on volume of each protein. VO was determined using Dextran Blue (Sigma; approximate molecular welght 2.000 KDa) Fl~ures 2 and 3 show the results of the gel flltratlon prote~n absorpt~on at 280 nm on the ord~nate- be~ng plotted against elution volume on the abscissa. It will be seen from Figure 2 that peak 2A is resolved into three separated peaks and a shoulder downstream of the first main peak. The components of m.w. 7 and 4 KDa were assigned .o the first main peak and its shoulder respectively. The 7 ~Da component is clearly the main one of peak 2A. Peak ci~ was resolvPd into three eparated peaks and a shoulder upstream of the main peak. the COmDOnentS of m.w.
7 and 4 KDa l)eino assicned t^ the shoulder and main pea~
respectively. The molecular weiallt5 of the principal components ; were determined to be about 7 and 4 KDa. (An attempt at m.w determination by SDS-PAGE gave anomalous results: it appears th~t 35 high molecular weight aggregates ^orm). The unexpectedly low ~

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molecular weights can probably be explained by clogging of the nominally 10,000 Dalton filter during ultrafiltration, allowing -smaller molecules to be retained. -S. Isoelectric focussina of cow whev ~roteins 05Isoelectric focussing was performed in tube gels (diameter, 4mm; length 11.5 cm). 4X polyacrylamide gels were prepared essentially as described by P.H. O Farrel1, J. B~ol. Chem. 250, 4007-4021 (1975) us~ng a mixture of amphollnes (4% v/v p~ range 5-8; 1% v/v pH range 3.5-10; BioRad), which gave a llnear 10gradient in the range 4.0-9Ø Samples (25 ~9 of the peak 2 protein) were dissolved ln a solution containing 9.5M urea, 2%
(w/v) NP40, l.6% (v/v) pH 5-8 ampholines and 0.4% (v/v) pH 3.5-10 ampholines. The anodic and cathodic solutions were lO mM H3P04 and 20 mM NaOH respectively. Electrophoresis was at 300 V for 1518 h, followed by 400 V for 4 h. Gels were extruded and fixed . first in 25% (v/v) isopropanol/lOX ~v/v) acetic acid, then in 5X
(wlv) TCA/57. (w/v) sulphosalicylic ac~d/1% (v/v) methanol, and were stalned 25Z (v/v) lsopropanol/lOX ~v1v) acetic acid containing 0.1~ (w/v) Coomassie Blue. Destaining was in ~sopropanol/acetic acid.
Two bands were obtalned. The maJor band has an isoelectrlc point of 4.84; the mlnor band focusses at an isoelectrlc polnt of 4.92.

6. SeDaration of Deak 2A comDonents bv chromatofocussinq 25Chromatofocussing separates proteins on the basis of their isoelectric point (pI). Resolution with the Pharmacia Mono P HR
5/20 column is such that molecules differing in pI by only 0.02 pH units can be separated. Mono P is a weak anion exchanger.
based on monobeads, i.e. monodisperse hydrophilic polymer particles (10 t 0;5 ~m diameter) into which various tertiary (-N+HR~ and quaternary (-N+R3) amine groups are introduced.
Mono P has a buffering capacity and the amount of charge it carries will vary with pH. Consequently, its ionic capacity will -also vary with pH. In chromatofocussing, a pH gradient is formed on the column by equilibrating it with start buffer and eluting - . . ~ -, :: . .

2~68533 12 -, with another buffer which is added in increasing amounts, thereby adjusting the solution progressively to a lower pH. Proteins bound to the column at the starting pH are eluted at different points on the pH gradient according to their pI.
05 A "Mono P" column was pre-washed with l ml of l M NaOH, and the equilibrated in lO mM imidazole, pH 6.5. The sample, 50~g of ultrafiltered bovine whey protein, peak ZA, obtained by anion-exchange chromatography as descr~bed ln sectlon 2 c!f th~s Example, was applled once the pH of the column eluant had returned to 6.5. The column was eluted with a l/lO v/v dilution of "Polybuffer" 74, pH 4.0 in distilled water. "Polybuffer"
(Pharmacia) contains numerous amphoteric buffering substances of dlfferent pKa. Buffers were filtered through 0.2 ~m filters and -degassed before use. Elution was carried out at a flow rate of O.S ml/min, until the pH of the eluant was 4.5. Protein eluting from the column was detected by monitoring absorbance of the eluant at 280 nm. ' :
Peak 2A eluted as three ma~or ~2A.l, 2A,2 and 2A.6) and three m1nor peaks ~2A.3, 2A.4 and 2A.S) as shown in Figure 4. The relative abundance of these components varies between separatlons. However, the separation is qual~tatively i!
reproducible in terms of th~ relatlve positlons of the peaks.
The second ma~or peak Peak 2A.2 whlch elutes at pH 5.0-4.9 is that whlch predomlnantly contalns the inhibitor ~see section 7 2S below).

7. Mammarv exDlant bioassav of chromatofocussed Deaks The bioassays were carried out as described in section 3.
The samples tested in this experiment had been fractionated by chromatofocussing of ion-exchange peak 2A as described in section S. They were dissolved in lOmM Hepe,. pH 7.4, at twice their concentration in the original mil~ and added to an equal volume of twice concentrated culture medium so as to be at 100-/o of their - -original milk concentration in normal strength culture medium.
Control cultures containing only the milk fraction diluent were included in each experlment. Average rates of lactose and caesin - ', ':

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W ~ 91/07439 PCT/GB90/01743 2 0.6~8S.3:3 synthesis were measured as described previously (section 3).
The amount of radioactive material was expressed as a percentage of that produced by the explants to which no milk fraction was added. The results are shown in Table 3 below. The 05 figures in parenthesis are the numbers of experiments performed on the various peaks. Results are the mean ~ s.e.m. where three experiments were carried out.
,.

Peak Number Lactose Synthesis Caesin Synthesis (% of control~ (7. of control) No addition (control) 100 100 2A.1 142 (1) 107 (1 : ~ .
2A.2 66,8 ~ 11.1 (3) 67.0 ~ l9.S (3 2A.6 101.5 (2) 102.9 (2) .
from the above table it can be seen that of the fractions tested only Peak 2A.2 was ~nhibitory.

8. Gel-filtration chromatoqraDhv of Peak 2A.2 Gel-filtration of peak 2A.2 (section 7) was carried out using ; an FPLC chromatography system and a Superose 12 HR10/30" column (Pharmacia), as described in séction 4 for the anion-exchange peaks. Peak 2A.2 eluted as the sole protein-containing p.eak.
The other peaks contained no detectable protein and were of low molecular weight (~ lkDa). The molecular weight of Peak 2A.2 was calculated to be about 7.0 kDa which was the molecular weight estimated for anion-exchange peak 2A in section 4.
~ 9. Isoelectric focusinq of chromatofocussed Deak 2A.2 ; ~ 20 Isoelectric foc~using was performed using the Pharmacia ~ "PhastGel" electrophoresis system. The method used "PhastGEL IEF

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W O 91/07439 ~Cr/GB90/~1743 2~S33 - 14 _ 4-6.5 . PhastGel IEF media are homogeneous polyacrylamide gels containing Pharmalyte carrier ampholytes. Pharmalyte generates stable, linear pH gradients in the gels during electrophoresis, in this case in the pH range 4 to 6.5. Proteins OS migrate under an electric field, essentially unhindered by ihe porous gel, to a point in the pH gradient that corresponds to their pI (isoelectric point~.
The sample, l~g, of Peak 2A.2 (sectlon 6) whlch had been extensively dialysed agalnst dlstllled water was applied to one well of the gel, Pharmacia pI Calibration Kit proteins were applied to wells on both sides of the sample well. The resulting protein bands were visualised by staining with Coomassie blue after electrophoresis. Peak 2A.2 gave rise to a single protein band corresponding to pl 4.9-S-0-''.''`'..
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Claims (8)

1. A protein which inhibits milk secretion by lactating cows and which is present in the second significant peak ("2A") when a nominally 10-30 KDa fraction of the whey proteins of the milk is resolved on an anion exchange column of particles of monodisperse hydrophilic polymers having pendant -CH2N(CH3)3+ groups, the particle diameter being 10?0.5µm, using 10 mM imidazole buffer, pH 7.0 and a sodium chloride elution gradient.

2. A protein according to Claim 1 which has a molecular weight as determined by gel filtration chromatography of about 7 KDa.

3. A protein according to Claim 1 or 2 which has an isoelectric point as determined in a tube of polyacrylamide gel within the range 4.8 to 4.95.

4. A protein according to Claim 1, 2 or 3, which is present in the second significant peak obtained when peak 2A is further resolved on a chromatofocussing column containing particles of monodisperse hydrophilic polymers having pendant tertiary (-N+HR2) and quaternary (-N+R3) amine groups where R represents an organic group the particle diameter being 10?0.5µm, using 10mM imidazole, pH 6.5 and amphoteric buffer of pH 4.0 to create a pH gradient of 6.5-4Ø

5. A protein according to claim 1, 2, 3 or 4 in unglycosylated form.

6. Antibody to a protein according to claim 1, 2, 3, 4 or 5.

7. A protein according to claim 1, 2, 3, 4 or 5 or which is generally similar thereto or antibody to such a protein, for regulating the lactation of animals.

8. A protein according to claim 7 wherein said animals are cows.