This report of the American Dairy Science Association Committee on the Nomenclature, Classification, and Methodology of Milk Proteins reviews changes in the nomenclature of milk proteins necessitated by recent advances of our knowledge of milk proteins. Identification of major caseins and whey proteins continues to be based upon their primary structures. Nomenclature of the immunoglobulins consistent with new international standards has been developed, and all bovine immunoglobulins have been characterized at the molecular level. Other significant findings related to nomenclature and protein methodology are elucidation of several new genetic variants of the major milk proteins, establishment by sequencing techniques and sequence alignment of the bovine caseins and whey proteins as the reference point for the nomenclature of all homologous milk proteins, completion of crystallographic studies for major whey proteins, and advances in the study of lactoferrin, allowing it to be added to the list of fully characterized milk proteins.

Exogenous daily oxytocin injections given immediately before milking increase milk production. To investigate the mechanism by which oxytocin increases milk production, oxytocin injections were given before and after milking, and saline injection was given before milking as a control. The experimental design was a replicated Latin square; two complete trials were performed: one with 12 cows (45 d) and another with 15 cows (95 d). In the first trial, the least squares means of milk production were 29.2, 29.3, and 28.3 kg for oxytocin injection before milking, oxytocin injection after milking, and saline injection before milking, respectively. In the second trial, the least squares means of milk production were 33.3, 32.9, and 32.4 kg for oxytocin injection before milking, oxytocin injection after milking, and saline injection before milking, respectively. Oxytocin before and after milking significantly increased milk production by 3%. The results suggest that increases in milk production may not be caused by removal of residual milk but by increased gland output of milk. The effect on milk plasmin activity, fat, protein, SCC, and lactose was nonsignificant and may indicate that effect of oxytocin is not manifested through an effect on cell remodeling.

High production of milk and its components are necessary to allow maximal growth of developing pigs. In this study, transgenic pigs were produced containing the alpha-lactalbumin gene, whose product is a potential limiting component in the production of milk. Two lines of transgenic pigs were produced to analyze the effects that overproduction of the milk protein alpha-lactalbumin may have on milk production and piglet growth. Transgenic pigs were produced through microinjection of the bovine alpha-lactalbumin gene. The gene construct contained 2.0 kb of 5' flanking region, the 2.0 kb coding region, and 329 bp of 3' flanking region. Sows hemizygous for the transgene produced as much as .9 g of bovine alpha-lactalbumin per liter of pig milk. The production of the bovine protein caused approximately a 50% increase in the total alpha-lactalbumin concentration of pig milk throughout a lactation. The concentration of bovine alpha-lactalbumin was highest on d 0 and 5 of lactation and decreased as lactation progressed. The ratio of bovine to porcine alpha-lactalbumin changed during the sow's lactation. This ratio was 4.3 to 1 on d 0 of lactation, but by d 20 of lactation the ratio was .43 to 1. This suggested that the bovine transgene and the endogenous porcine gene are under slightly different control mechanisms. The higher level of total alpha-lactalbumin present on d 0 of lactation was correlated with higher lactose percentage on d 0 in transgenic sows (3.8%), compared with controls (2.6%) (P < .01). Although there was also a trend for higher lactose percentage in transgenic sows on d 5 and 10 of lactation, no significant differences were observed. These data suggest that alpha-lactalbumin is limiting early in lactation of swine. Furthermore, higher concentrations of alpha-lactalbumin early in lactation may boost milk output.

BackgroundThe ability to site-specifically conjugate a protein to a payload of interest (e.g., a fluorophore, small molecule pharmacophore, oligonucleotide, or other protein) has found widespread application in basic research and drug development. For example, antibody-drug conjugates represent a class of biotherapeutics that couple the targeting specificity of an antibody with the chemotherapeutic potency of a small molecule drug. While first generation antibody-drug conjugates (ADCs) used random conjugation approaches, next-generation ADCs are employing site-specific conjugation. A facile way to generate site-specific protein conjugates is via the aldehyde tag technology, where a five amino acid consensus sequence (CXPXR) is genetically encoded into the protein of interest at the desired location. During protein expression, the Cys residue within this consensus sequence can be recognized by ectopically-expressed formylglycine generating enzyme (FGE), which converts the Cys to a formylglycine (fGly) residue. The latter bears an aldehyde functional group that serves as a chemical handle for subsequent conjugation.ResultsThe yield of Cys conversion to fGly during protein production can be variable and is highly dependent on culture conditions. We set out to achieve consistently high yields by modulating culture conditions to maximize FGE activity within the cell. We recently showed that FGE is a copper-dependent oxidase that binds copper in a stoichiometric fashion and uses it to activate oxygen, driving enzymatic turnover. Building upon that work, here we show that by supplementing cell culture media with copper we can routinely reach high yields of highly converted protein. We demonstrate that cells incorporate copper from the media into FGE, which results in increased specific activity of the enzyme. The amount of copper required is compatible with large scale cell culture, as demonstrated in fed-batch cell cultures with antibody titers of 5 g · L−1, specific cellular production rates of 75 pg · cell−1 · d−1, and fGly conversion yields of 95–98 %.ConclusionsWe describe a process with a high yield of site-specific formylglycine (fGly) generation during monoclonal antibody production in CHO cells. The conversion of Cys to fGly depends upon the activity of FGE, which can be ensured by supplementing the culture media with 50 uM copper(II) sulfate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0254-0) contains supplementary material, which is available to authorized users.

The goal of this study was to determine whether the presence of the bovine alpha-lactalbumin transgene in first-lactation gilts enhances lactational performance and litter growth. Transgenic and sibling nontransgenic gilts were bred to nontransgenic boars. Litters were standardized to 10 piglets within 24 h of farrowing. Milk production was measured by the weigh-suckle-weigh method on d 3, 6, 9, and 12 of lactation. Bovine alpha-lactalbumin was present in the colostrum and milk of transgenic gilts throughout lactation. The expression of the transgene was associated with alterations in composition of mammary secretions, especially in early lactation. Lactose concentrations were greater (P < 0.05) in mammary secretions of transgenic gilts during the first 12 h postpartum compared with controls. In contrast, total solids concentration in mammary secretions from transgenic gilts were lower (P < 0.05) relative to controls during the first 6 h postpartum. Transgenic gilts produced more milk than controls on d 3, 6, and 9 of lactation (P < 0.01). By d 12, differences in milk production between transgenic and control sows were no longer different. Lactose intake by transgenic-reared litters was greater than lactose intake by control-reared litters on d 6 of lactation (P < 0.05). Total solids intake was significantly greater (P < 0.05) by transgenic-reared litters on d 3 and 6 compared to control-reared litters. The day x genotype interaction on litter weight gain after birth was highly significant (P = 0.011), with transgenic-reared litters gaining weight at a greater rate than control-reared piglets. Expression of the transgene was associated with increased milk production in lactating gilts and increased growth of transgenic-reared piglets. Increased lactose synthesis in response to the presence of the transgene may result in increased milk production in early lactation, leading to increased milk component intake by transgenic litters, and ultimately to increased growth of litters reared by first-parity transgenic gilts.

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The alpha-lactalbumin (+15) polymorphism (a single base variation 15 basepairs 3' of the alpha-lactalbumin transcription start point) was examined for its usefulness as a genetic marker for Holsteins. The +15 polymorphism is located in a region of the gene that is potentially involved in the regulation of alpha-lactalbumin gene expression. Animals from two dairy herds and young sires from progeny-testing programs of four AI organizations were used in the analysis. A group of sons from a heterozygous sire were also evaluated. Each individual animal was genotyped at the alpha-lactalbumin (+15) locus, and differences of genotypes were investigated. Estimated differences among alleles were calculated for PTA for milk, kilograms of protein, protein percentage, protein dollars, kilograms of fat, fat percentage, and fat dollars. Animals having the alpha-lactalbumin (+15) AA (an adenine on both alleles at position +15) genotype had statistically higher PTA for milk, kilograms of protein, protein dollars, kilograms of fat, and fat dollars than did the alpha-lactalbumin (+15) BB (a cytosine, guanine, or thymine on both alleles at position +15) animals. The alpha-lactalbumin (+15) BB animals had higher protein and fat percentages than the alpha-lactalbumin (+15) AA animals. Animals that were heterozygous at this locus, alpha-lactalbumin (+15) AB, had intermediate values for all traits analyzed. These results indicate a potential marker or actual locus effect of the alpha-lactalbumin (+15) polymorphism in Holstein cattle.

Lactose synthase (a complex of beta1,4-galactosyltransferase and alpha-lactalbumin) forms lactose in the Golgi complex of mammary epithelial cells. To determine whether alpha-lactalbumin is a limiting component in this complex, transgenic mice that expressed bovine alpha-lactalbumin were studied. Transgenic mice produced 0.5 to 1.5 mg/ml of bovine alpha-lactalbumin in their milk, 5- to 15-fold more alpha-lactalbumin than in milk of control mice. Transgenic and control mice produced milk with the same concentrations of lactose, cream, and total solids, and showed similar mammary gland growth, morphology, and histology. Milk from transgenic mice had 0.6% less protein than milk from control mice (P < 0.05). The in vitro lactose synthase activity in mammary gland homogenates from alpha-lactalbumin transgenic mice was increased (P < 0.05), demonstrating that bovine alpha-lactalbumin could interact with murine beta1,4-galactosyltransferase. Pups reared by lactating transgenic mice showed a 4% increase in growth on d 10 of lactation, suggesting that milk production was increased (P = 0.06). Milk volume, estimated using the weigh-suckle-weigh technique, tended to be higher (although not significantly) in transgenic mice (P = 0.11). These results suggest that augmenting alpha-lactalbumin expression in the dam increases the growth of suckling offspring.