J. Anim. Sci. 2002. 80:1863-1870
© 2002 American Society of Animal Science
Purification of porcine ß-casein, N-terminal sequence, quantification in mastitic milk1,2
A. C. W. Kauf and
R. S. Kensinger3
Department of Dairy and Animal Science, Pennsylvania State University, University Park 16802
3 Correspondence:
phone: 814-863-0558; fax: 814-865-7442; E-mail:
RSK7{at}psu.edu.
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Abstract
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The objectives of the study were to purify porcine ß-casein from sow’s milk, to determine N-terminal amino acid sequence, to develop specific antisera against porcine ß-casein, and to use that antisera to evaluate milk samples from a mastitis study. Milk was collected by hand milking a Yorkshire by Duroc crossbred sow following oxytocin administration on d 27 of lactation. A casein-enriched fraction was then prepared by iso-electric precipitation. Porcine ß-casein was then purified by liquid chromatography on a Mono Q anion-exchange column, and checked for purity with SDS-PAGE. An apparent molecular weight of 29,000 Da was estimated from SDS-PAGE. N-Terminal amino acid sequence was determined by Edman degradation to be RAKEELNASGETVE. Rabbits (n = 2) were immunized with ß-casein mixed with Freund’s complete (primary) or incomplete (boosters) adjuvant at 4-wk intervals. Antiserum collected from one rabbit 112 d after primary immunization detected 30 to 100 ng ß-casein by Western blot procedure when used at a dilution of 1:2 x 106. The antiserum was specific for porcine ß-casein, but showed some cross-reactivity with equine casein. It was determined by Western blot procedure that mammary inflammation induced by lipopolysaccharide infusion resulted in a 41% decrease in the ß-casein concentration of sow milk.
Key Words: Amino Acid Sequences • Antiserum • ß-Casein • Liquid Chromatography • Mastitis • Pigs
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Introduction
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Milk production by most sows restricts the genetic potential for lean-tissue growth of modern piglets (Boyd et al., 1995). Furthermore, the pre-weaning mortality rate of piglets continues to represent a significant economic loss (Straw et al., 1998). Mastitis in lactating sows, also known as porcine hypogalactia syndrome (PHS), can reduce milk consumption by piglets, increase mortality, and reduce growth (Curtis, 1974; Dyck et al., 1987).
The above observations suggest that improved sow-milk-solids yield should enhance litter performance. Previous studies in our laboratory examined the regulation of lactogenesis (Jerry et al., 1989); protein synthesis (San Gabriel et al., 1994); and ß-casein gene expression (Cao et al., 1995; Kensinger et al., 1996) in the sow. An antiserum capable of specifically detecting porcine ß-casein would be very useful to quantify secreted ß-casein in milk or culture media. In order to make one, it was necessary to purify porcine ß-casein from milk, as neither the protein nor an antiserum against it is currently available commercially. There are reports on the purification of porcine caseins (Mulvihill and Fox, 1979; Cerning-Beroard and Zevaco, 1984; Erhardt, 1989), but these methods were deemed insufficient for our needs. A goal of this project was to purify milligram quantities of porcine ß-casein in order to provide protein for assay development. An antiserum developed in the course of this study was used to evaluate milk samples from an endotoxin challenge model of mastitis (Kensinger et al., 1999).
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Materials and Methods
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Milk Donor for ß-casein Purification
Milk (1,200 mL) from a single sow was used for the purification of porcine ß-casein. It was collected by manual expression following oxytocin administration (10 IU i.m.) from a Yorkshire by Duroc crossbred sow on d 27 of lactation after an overnight separation of piglets from the sow.
Determination of Protein Concentration
Protein concentrations in solutions during casein purification were determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Protein concentrations of milk samples were determined by a modification of the Lowry assay (Lowry et al., 1951), using saponification in 1 M NaOH and absorbance at 750 nm.
Electrophoresis Techniques
The SDS-PAGE, a modification of Laemmli (1970), was used for analysis of samples using a Mini-Protean 3 gel electrophoresis apparatus (Bio-Rad, Hercules, CA). Separating gels were composed of 15% acrylamide or 13% acrylamide with 4 M urea, 0.01% SDS, and 0.375 M tris at a final pH of 8.8. Stacking gels were composed of 4% acrylamide or 4% acrylamide with 4 M urea, 0.01% SDS, and 0.125 M tris at a final pH of 6.8. Gels were polymerized using 0.01% ammonium persulfate (APS) and 0.001% N,N,N',N'-tetramethylethylenediamine (TEMED). A tris-glycine-SDS buffer (0.049 M tris, 0.366 M glycine, and 0.1% SDS, pH 8.3) was used for electrophoresis. Samples were added to 15 µL sample buffer (0.12 M tris, 3.84% SDS, 19.2% glycerol, 9.6% ß-mercaptoethanol, and 0.024% bromophenol blue, pH 6.8), brought to a final volume of 40 µL, and heated for 8 min in boiling water prior to loading. Current was then applied at a constant voltage of 58 V until samples had entered the separating gel; voltage was then increased to 190 V until completion. Gels were stained for 10 min with 0.1% R250 Coomassie brilliant blue in 10% acetic acid and 40% methanol. Gels were destained overnight in 10% acetic acid and 7% methanol with two buffer changes. Images of gels were captured using the Eagle Eye still-video imaging system (Stratagene, La Jolla, CA).
Purification of Porcine ß-casein
Porcine ß-casein was purified from a sample of porcine milk by a combination of defatting, precipitation at pH 4.6, and anion-exchange liquid chromatography (LC). Milk was defatted by centrifugation (5,000 x g, 15 min), and the casein fraction precipitated by incubation at pH 4.6. A casein-enriched pellet was isolated by centrifugation (5,860 x g, 60 min), washed three times with distilled water, lyophilized, and stored at -20°C until use. This precipitated casein is referred to as the whole casein fraction (Wc).
The Wc was further fractionated by LC on a 5-mL Mono Q anion-exchange column using a Biologic LP system (Bio-Rad). The Wc was dissolved in chromatography buffer (4 M urea, 0.01 M imidazole, pH 6.75) to a concentration of 25 mg protein/mL and stored overnight at 4°C. The Wc sample (37.5 mg protein) was applied to the column with chromatography buffer, and caseins were eluted at a flow rate of 1 mL/min. Caseins were eluted with a step-wise gradient of NaCl in chromatography buffer: 5 mL at 50 mM, 15 mL at 95 mM, 15 mL at 165 mM; 15 mL at 205 mM, 10 mL at 290 mM; and 10 mL at 500 mM NaCl. Fractions (n = 85, 1 mL each) were collected and characterized by SDS-PAGE as described previously. The optimized chromatographic procedure was performed five times.
Electrophoretic Transfer
Purified porcine ß-casein to be used for N-terminal amino acid sequence analysis was electro-blotted onto polyvinylidene difluoride (PVDF) membrane (Pro-Blott, Bio-Rad) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). Samples were fractionated by SDS-PAGE (15% acrylamide) under reducing conditions. Transfer was performed in 4°C transfer buffer (10 mM 3-cyclohexylamino-1-propanesulfonic acid, 10% methanol, at pH 11) at a constant 50 V for 2 h.
Milk samples to be used for Western blotting were electro-blotted onto nitrocellulose membrane (0.45 µm; Hybond-C, Amersham-Pharmacia Biotech, Piscataway, NJ) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). Samples were isolated by SDS-PAGE (13% acrylamide with 4 M urea) under reducing conditions. Transfer was performed in 4°C transfer buffer (0.425 M tris, 0.192 M glycine, 20% methanol, pH of 8.3) at a constant 350 mA for 1.5 h.
Amino Acid Sequence Determination
After the purification of porcine ß-casein, the sample was sent to the Macromolecular Core facility (PSU) for N-terminal amino acid sequencing. Sequence was obtained by automated Edman degradation followed by HPLC and UV detection (Edman and Begg, 1967), using an Applied Biosystems 477A protein sequencer (Applied Biosystems, Foster City, CA).
Antisera Generation
Porcine ß-casein in PBS was brought to a final concentration of 1 mg/mL. Antisera generation was performed by a commercial laboratory using a modification of Vaitukaitis et al. (1971). Primary injections (400 µg purified porcine ß-casein in PBS mixed with complete Freund’s adjuvant) were administered to two New Zealand White rabbits, and booster injections (200 µg of ß-casein added to incomplete Freund’s adjuvant) were delivered subcutaneously on d 28, 56, and 84 following primary injection. Blood was collected from each rabbit prior to immunization, at 2 wk following each booster injection, and a final bleeding was performed 112 d following primary injection.
Western Blotting
Western blotting was performed at room temperature using the Vectastain ABC and DAB substrate kits (Vector Labs, Burlingame, CA). The ABC kit instructions were modified to optimize Western blotting for porcine ß-casein and milk samples. Initial tests of antisera dilution were performed in 15-mL conical tubes; all other milk-sample testing was performed in 50-mL conical tubes, with gentle agitation using a LabQuake shaker (Barnstead/Thermolyne: Dubuque, IA). Nitrocellulose membranes were first equilibrated in modified TTBS (0.5% Tween-20, 0.1 M tris, 0.9% NaCl, pH 7.5) for 30 min following electrophoretic transfer. Membranes were then transferred to a solution of rabbit anti-porcine ß-casein antisera (1:2 x 106 dilution in modified TTBS) for 30 min, followed by washing in modified TTBS with four changes over a total of 25 min. Antisera dilutions were initially tested over a range from 1:100 to 1:2 x 106. Membranes were then transferred for 30 min to a solution of biotinylated goat anti-rabbit antibody (0.5% antibody, 1% normal goat sera, and 1% normal gilt sera in modified TTBS) prepared 1 h previously, followed by washing in modified TTBS with four changes over a total of 25 min. Membranes were then transferred to a solution of avidin-biotinylated horseradish peroxidase (ABC reagent in modified TTBS) for 30 min, followed by washing in modified TTBS with four changes over a total of 25 min. Finally, membranes were transferred to a solution of DAB substrate in distilled water for 20 min, and then transferred to distilled water for 5 min and allowed to air-dry. Images of Western blots were captured using the Eagle Eye (Stratagene, La Jolla, CA) still-video imaging system and manipulated as needed, using Photoshop (Adobe software, San Jose, CA) prior to optical density analysis using ONE-Dscan (Scanalytics Corporation, Fairfax, VA). Optical densities were determined in triplicate, and the mean was used for subsequent statistical analysis.
Antisera Specificity Testing
Milk samples (2 to 50 mL) used for antisera specificity testing were collected by hand milking various species on Penn State University farms. Bovine (n = 2, Holstein), ovine (n = 2, Dorset), and equine (n = 3, Quarterhorse) samples were all collected after the wk 1 of lactation, and were therefore considered to be mature milk.
Endotoxin Challenge Mastitis Model
Milk samples (3 to 5 mL) to be used for Western blot analysis were obtained from primiparous Yorkshire (n = 6) and 3/4 Yorkshire, 1/4 Hampshire (n = 1) gilts that were subjected to intramammary endotoxin challenge (Kensinger et al., 1999). On experimental days, two functioning and not previously infused mammary glands in each gilt were infused with endotoxin (1.5 µg/kg BW) at 0700. Milk samples were collected between 0900 and 1200 by manual expression during one or more nursing episodes from both treated and control mammary glands within each gilt on each day. Endotoxin administration and milk-sample collection were repeated on alternating days for 3 d, typically on d 3, 5, and 7 of lactation. Consequently, 21 milk samples each from both control and mastitic glands were obtained. This experiment was reviewed and approved by the Penn State Institutional Animal Care and Use Committee (99R016-0), as well as by the Penn State University Biosafety Committee (UBC 99/17).
Testing of Sow Milk Samples
Milk samples were collected from gilts on each of 3 d from both endotoxin-infused and control mammary glands. Milk samples were analyzed by Western blot technique, and the blots were analyzed for optical density. A 250-ng sample purified porcine ß-casein was used as a positive control on one lane of each blot. Optical density analysis of Western blots was used to determine milk ß-casein content per 10 µg of total milk protein.
Statistical Analysis
The endotoxin challenge study utilized a replicated randomized complete block design with samples from a total of seven animals. Treatment represented endotoxin infusion vs control mammary glands. Data (total milk protein as determined by Lowry protein assay or milk ß-casein concentration as determined by optical density analysis of Western blots) were analyzed using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC) with gilt, day, and treatment as class variables. Factors in the statistical model included gilt, day, and day x treatment interaction. The Proc Means procedure of SAS (SAS Inst. Inc.) was used to generate means for presentation, and statistical significance was declared at a probability level of 
0.05.
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Results
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Chromatography
The fractionation of porcine caseins resulted in five A280 absorbance peaks, and a separation of the casein subspecies (Figure 1
). Peak 1 represents proteins that bind weakly to the µN+(CH3)3 functional group, and so elute in the breakthrough (Figure 1). This peak was largely composed of very small (<14,000 Da) proteins and/or protein fragments found in very low concentrations. Peak 2, eluted by 95 mM NaCl, was largely composed of 
-casein (Figure 1). Peak 3, eluted by 165 mM NaCl, contained primarily ß-casein (Figure 1). However, 
s2-casein also begins to elute in the latter half of this peak. The purest ß-casein was found in fractions 33 to 37, being eluted by the initial 5 mL of 165 mM NaCl. Peak 4, eluted by 205 mM NaCl, contained a mixture of s1, s2, and -casein (Figure 1). The majority of the remaining protein and protein fragments were eluted by 290 mM NaCl (peak 5, Figure 1). A small amount of residual protein was eluted by 500 mM NaCl, confirming that the majority of protein was eluted from the column matrix.
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Figure 1. Chromatogram of casein fractionation by anion-exchange liquid chromatography on 5-mL Mono Q column. Porcine whole casein (37.5 mg in 1.5 mL of 4 M urea, 0.01 M imidazole, pH 6.75) was loaded and eluted at a 1 mL/min flow rate by a NaCl gradient of 0 to 0.5 M. The solid line represents the NaCl gradient (mS conductivity), and the dashed line represents protein concentration of fractions (A280). The highest purity ß-casein was recovered from peak 3, fractions 33 to 37.
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Replicated Purification to make ß-casein Pool
A quantity of 5 to 6 mg purified porcine ß-casein was needed for N-terminal amino acid sequence determination, polyclonal antisera generation, and Western blotting. By twice performing five replicates of the entire chromatographic procedure, two pools of purified porcine ß-casein were created. Pools consisted of fractions 33 to 37 from each replicate, and all fractions were subjected to analysis by SDS-PAGE in 4 M urea under reducing conditions to confirm purity before being pooled (data not shown). The creation of these pools resulted in more than 9 mg of purified porcine ß-casein. The purity of the pools was confirmed by SDS-PAGE analysis (Figure 2) prior to sampling for N-terminal amino acid sequence determination. Samples were also used for generation of polyclonal antisera. SDS-PAGE analysis revealed the presence of minimal contaminants, as no significantly larger or smaller molecular weight proteins than the putative ß-casein were revealed (Figure 2).
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Figure 2. SDS-PAGE analysis of purified porcine ß-casein. Mr = molecular weight standards in daltons. Lane A = 10 µg whole casein fraction. Lanes B to E = 5, 10, 15, and 20 µg, respectively, purified porcine ß-casein from pool 1. Lanes F to I = 5, 10 15, and 20 µg, respectively, purified porcine ß-casein from pool 2.
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Porcine milk proteins were compared to whole casein fraction, porcine whey, and bovine 
-globulins by SDS-PAGE (data not shown). The protein putatively identified as porcine ß-casein migrated like the dominant protein band in both mature milk and whole casein fraction, at an apparent molecular weight of 29,000 Da (see Figure 3).
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Figure 3. SDS-PAGE analysis of mature sow milk (Lane A), whole casein fraction (Lane Wc), and purified porcine ß-casein (Lane C). Mr = molecular weight standards in daltons. Lanes A, Wc, and C have 10, 10, and 1 µg protein, respectively.
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N-terminal Amino Acid Sequence
Edman degradation (15 cycles) was performed to determine N-terminal amino acid sequence of the purified ß-casein. The resultant amino acid sequence, beginning with the N-terminus, was RAKEELNASGETVE, corresponding to Arg-Ala-Lys-Glu-Glu-Leu-Asn-Ala-Ser-Gly-Glu-Thr-Val-Glu.
Effectiveness of Purification
In order to confirm sequential enrichment of porcine ß-casein, the purified porcine ß-casein sample was compared with mature sow milk and to the Wc (Figure 3
). It was determined that purified porcine ß-casein was greater than 95% pure (Figure 3). Sow milk protein is approximately 54% casein (Aimutis et al., 1982; Klobasa et al., 1987), and the ratio of ß- to s-caseins is 2.3:1 (O’Connor and Fox, 1973). If -casein represents 10% of total caseins (Figure 3), then ß-casein represents 63% of total caseins, and approximately 34% of sow-milk protein. Thus, enrichment of ß-casein in Lane C (Figure 3) represents at least a threefold enrichment over the concentration in sow milk.
Antisera Titering for use with Western Blots
The optimal dilution of antiserum to detect ß-casein in 10 µg protein from a mature sow-milk pool was determined to be 1:2 x 106 (data not shown). Sera collected from the terminal bleeds (112 d after primary immunization) of both rabbits were similar in specificity at this and all dilutions tested (1:100 to 1:2 x 106). Sera collected from rabbits prior to antisera generation had no antibodies (data not shown). A clear induction of antibodies specific for ß-casein was achieved 14 d after the first booster, 42 d after primary injection (data not shown). The density of the ß-casein band continued to improve slightly until the terminal bleed 112 d after primary injection (data not shown). Antiserum from rabbit 918 was selected for subsequent use, as it was slightly superior to that from rabbit 919, as indicated by a denser band for ß-casein and less background staining (data not shown). When used at a dilution of 1:2 x 106, this antiserum collected 112 d after primary immunization detected 30 to 100 ng of porcine ß-casein by Western blot procedure (Figure 4), which would be the equivalent of the mass of ß-casein found in 4 to 14 nL of mature sow milk. The presence of bands smaller than ß-casein (Lanes E to G, Figure 4) may be due to genetic polymorphism or nonspecific cleavage fragments of ß-casein, and was not considered a problem given the very high mass of ß-casein in those lanes.
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Figure 4. Western blot analysis of polyclonal antiserum collected from rabbit 918 on d 112 after primary immunization and used at a 1:2 x 106 dilution. Mr = pre-stained molecular weight standards in daltons. Lanes A to F = 30, 100, 300, 500, 1250, and 2500 ng porcine ß-casein. Lane G = 10 µg whole sow-milk protein (d 8 to 10 pool).
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Specificity of Antisera
Antiserum from rabbit 918 had very strong specificity for porcine ß-casein (Lane G, Figure 4; Lanes A to B, Figure 5). Antiserum readily detected ß-casein in mature porcine milk, but detected no ß-casein in either bovine or ovine milk, even though the gel on the left showed abundant bovine and ovine caseins (Figure 5). There was a very minor cross-reactivity with ß-casein in equine milk, as seen by the faint bands in lanes G and H (Figure 5).
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Figure 5. SDS-PAGE (left) and Western blot (right) analysis of milk protein from porcine, bovine, ovine, and equine. Mr (left) = molecular weight standards in daltons. Mr (right) = pre-stained molecular weight standards in daltons. Lanes A, B = sow (x 1 to 1, x 1 to 3) milk from d 8 and 10. Lanes C, D = cow (334, 335) milk from d 21 of lactation. Lanes E, F = ewe (98158, 99158) milk from d 28 and 17 of lactation. Lanes G, H = horse (Lady, Stanza) milk from d 13 and 11 of lactation. All lanes contain 10 µg total milk protein.
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Effect of Mastitis on Protein Content of Sow Milk
Protein concentrations in milk for all samples from endotoxin-infused and control glands averaged 6.27 and 6.48% (±0.22), respectively, which were not different. The main effects of gilt and day were also not significant (Table 1). Endotoxin infusion decreased total milk-protein concentration on d 3, but not on d 5 and 7 (Figure 6). Thus, a significant day x treatment interaction on milk-protein concentration was detected (Table 1, Pr < 0.01).
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Table 1. Analysis of variance of effect of endotoxin infusion upon total milk protein or ß-casein concentration. Data included observations (42 samples) from seven animals from d 3 through 8 of lactationa
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Figure 6. Milk-protein response to endotoxin infusion on d 3, 5, or 7 of lactation in the sow. Milk from control glands indicated by solid bar, and milk from inflamed glands indicated by light bar. Error bars are ± 0.38%.
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Effect of Mastitis on ß-casein Content of Sow Milk
The ß-casein concentrations per 10 µg of total milk protein from endotoxin-infused and control glands averaged 208 and 354 ng (±22.59), respectively. The difference (41% less ß-casein as a result of mammary inflammation) was highly significant, as indicated by the main effect of treatment (Table 1, Pr < 0.01). There were also significant differences between gilts in mean ß-casein content (Table 1, Pr < 0.01). Endotoxin infusion appeared to have the greatest effect on ß-casein on d 3 (Figure 7), but ß-casein content did not significantly differ among days (Table 1). There was no day x treatment interaction (Table 1).
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Figure 7. ß-casein concentration in milk as affected by endotoxin infusion on d 3, 5, or 7 during early lactation in the sow. Milk from control glands indicated by solid bar, and milk from inflamed glands indicated by light bar. Error bars are ± 44.32 ng ß-casein/10 µg total milk protein.
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Discussion
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The procedure used in this study resulted in a highly purified sample of porcine ß-casein. The method used was a modification of the method of Davies and Law (1987) for the fractionation of bovine casein, but with a few key differences. Our method utilizes significantly more whole casein (37.5 mg vs 1.7 to 3.3 mg) at a higher concentration (25 mg/mL vs 5 mg/mL), allowing for the purification of a larger amount of ß-casein in a single replicate. Our method also utilizes a different buffer composition (4 M urea, 0.01 M imidazole, pH 6.75 vs 0.005 M bis-tris-propane, 3.3 M urea, pH 7.0) and does not require the alkylation of the whole casein prior to fractionation. It was deemed desirable to avoid alkylation, since it is not clear how it would affect the utility of subsequently developed rabbit antibodies.
The elution order of casein subspecies (-, ß-, s2-, then s1-casein) is identical between Davies and Law (1987) and the present study, although the concentration of NaCl required to elute each casein subspecies is lower with our method. This is probably a function of differences in amino acid sequence between the two proteins or net charge given that different pH levels were utilized.
Both the urea-imidazole method developed in this study and the Davies and Law (1987) method result in highly purified samples of ß-casein. While latter fractions (37 to 42) of peak 3 in our method showed co-elution of s2-casein (data not shown), the initial fractions (33 to 36) are very pure. A sufficient amount of highly enriched porcine ß-casein was recovered from these fractions that the amount of ß-casein lost in the latter fractions was deemed acceptable. The Davies and Law (1987) method yielded an equally pure sample of bovine ß-casein while recovering a greater percentage of that loaded. Anion-exchange chromatography (five replicates) with the urea-imidazole method resulted in the recovery of >9 mg (
1.8 mg per replicate) of highly purified porcine ß-casein.
The N-terminal amino acid reported in this study is also identical to the N-terminal amino acid (arginine) reported by Mulvihill and Fox (1979). The amino acid sequence of the 14 N-terminal amino acids (RAKEELNASGETVE) determined by Edman degradation for the purified porcine ß-casein sample reported in this study is also identical to the sequence of the 14 N-terminal amino acids predicted by Alexander and Beattie (1992) from the cDNA sequence for porcine ß-casein. Therefore, the protein purified by iso-electric precipitation and anion-exchange LC is authentic porcine ß-casein. This is the first verification of the 14 N-terminal amino acids predicted by Alexander and Beattie (1992). There are no reports of directly determined amino acid sequence for porcine ß-casein to verify the remainder of the sequence prediction (last GENBANK search, 2 April 2001).
The theoretical molecular weight of porcine ß-casein based upon predicted amino acid sequence was 24,397 Da (Alexander and Beattie, 1992), whereas Mulvihill and Fox (1979) reported a molecular weight of 24,900 Da based upon gel filtration chromatography. Our purified sample of porcine ß-casein migrated in SDS-PAGE as one major band at an apparent molecular weight of 29,000 Da, which was identical to that reported by Erhardt (1989). Numerous papers in the literature show that apparent molecular weights of caseins in SDS-PAGE are greater than predictions based on amino acid sequence.
A few minor bands of smaller apparent molecular weight were detected when the sample was subjected to SDS-PAGE under reducing conditions in 4 M urea (Figure 2). Dr. Harry Farrell (USDA Eastern Regional Laboratory) used our whole casein preparation for characterization of ß-casein by alternate methods and reported four bands on urea electrophoresis (personal communication). There are reports in the literature of polymorphism by charge and molecular weight in porcine ß-casein (Glasnak 1966, 1968a,b; Gerrits et al., 1969). The staining of bands at a smaller apparent molecular weight than ß-casein during Western blot procedure (Lane G, Figure 4) may also indicate polymorphisms in porcine ß-casein. Additionally, Hollar et al. (1991) reported the separation of genetic variants of bovine ß-casein, and polymorphism has been reported in the water buffalo ß-casein gene (Das et al., 2000; Klotz et al., 2000). Given the above information, the minor bands observed in the current study are likely variants due to phosphorylation, glycosylation or genetic sequence. However, that was not tested as a part of this study.
Antiserum obtained from the final bleed of one rabbit was selected as having the optimal titer and specificity, and was used at a very high dilution (1:2 x 106) to detect both purified porcine ß-casein and ß-casein in a sow-milk sample. This antiserum also showed very high species specificity (Figure 5), failing to detect bovine or ovine ß-casein, and showing only very minor cross-reactivity with equine ß-casein. Some cross-reactivity among different species was not surprising given the conservation among species.
Studies of mastitic milk in dairy cattle suggest that the loss in casein content is accompanied by increased immunoglobulins (Carroll and Jain, 1969), albumen, and lactoferrin (Shuster et al., 1991). Furthermore, the resulting inflammation in the endotoxin-infused glands leads to a significant reduction in milk secretion, as it was very difficult to hand milk the inflamed glands when compared to the healthy glands. The significance is that the piglets nursing the endotoxin-infused glands received less milk protein and less total protein than did their littermates nursing healthy glands.
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Implications
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This paper describes the purification and sequence analysis of porcine ß-casein. The purified casein was used to generate an antiserum that was used in Western blotting to quantify ß-casein in milk from sows. Results from analysis of milk from endotoxin-infused and control glands showed that ß-casein is reduced significantly in mastitic glands. Given the known value of caseins to the nutritional needs of the baby pig, this is hypothesized to be part of the mechanism leading to reduced growth rate by litters of affected sows. This ß-casein assay can be used in future studies to further our knowledge of the regulation of porcine milk protein secretion.
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Footnotes
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1 This work was supported by Hatch Project PSU-3420, as well as a grant from the Pennsylvania Department of Agriculture Animal Health Commission (Contract Me 448319). 
2 The authors would like to acknowledge the contributions of Ana San Gabriel, Ann Magliaro, Dawn Sanzotti, Rebecca Perri, Dante Pighetti, Emma Herscher, Sarah Reuss, Lori Cesario, and Stephanie Herr.
Received for publication August 7, 2001.
Accepted for publication February 6, 2002.
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Abstract
Introduction
