Expression and Characterization of Bioactive Recombinant Human α-Lactalbumin in the Milk of Transgenic Cloned Cows

Abstract

Improvement of the nutritional value of cow milk with transgenic expression of recombinant human alpha-lactalbumin (alpha-LA) has been previously attempted. However, the detailed characterization of the recombinant protein and analysis of the transgenic milk components are not explored yet. Here, we first report production of healthy transgenic cows by somatic cell nuclear transfer, in which expression of up to 1.55 g/L of recombinant human alpha-LA was achieved. The recombinant human alpha-LA was purified from transgenic milk and displayed physicochemical properties similar to its natural counterpart with respect to molecular weight, structure, and regulatory activity for beta-1,4-galactosyltransferase. Additionally, no N-glycosylation was found in the recombinant human alpha-LA, whereas the endogenous bovine alpha-LA was glycosylated at the unusual site (71)Asn-Ile-(73)Cys. Compared with milk from nontransgenic cows, expression of the transgene did not materially alter milk composition, such as fat and protein content. Our research thus provides scientific evidence supporting the feasibility of humanizing cow milk.

Comparative Proteomics of Milk Fat Globule Membrane Proteins from Transgenic Cloned Cattle

“…The TC-LF group expressed recombinant human lactoferrin at high levels, i.e., 2.5–3.8 g/l [25], the TC-LA group, expressed recombinant human α-lactalbumin at intermediate levels, i.e., 1.5 g/l [26], and the TC-LZ group expressed recombinant human lysozyme at low levels, i.e., 13–28 mg/l [24]. The expression profile of TC-LF group displayed a marked difference with that of other TC groups but the range of variation was comparable to the C group.…”

“…The three transgenic cattle lines TC-LZ (n = 10), TC-LA (n = 4), and TC-LF (n = 3) have been described [24]–[26] and were compared to cloned cattle (C, n = 3) which were also cloned by somatic cell nuclear transfer (SCNT), and conventionally bred cattle (N, n = 9) of a similar genetic background. The cattle were similar in age and lactation period and were housed under the same conditions.…”

“…We genetically engineered three transgenic bovine lines that specifically express recombinant human α-lactalbumin (TC-LA group), lactoferrin (TC-LF group), and lysozyme (TC-LZ group) [24]–[26], from which the compositions of the milk include the human proteins with higher expression level compared to that from conventionally bred animals. Prior research for milk whey proteins and the nutritional components from transgenic cloned cattle have indicated that the expression of exogenous proteins did not significantly change the milk whey protein profile, and the mean values for the majority of the measured parameters were within the normal range [27].…”

Production of Transgenic Bovine Cloned Embryos Using Piggybac Transposition

“…Even though transgenesis is not well investigated in cattle compared to mice, transgenic cattle embryos produced with well-established in vitro culture systems have been used for propagating elite cows [16], producing disease resistant cattle [17,23] or gene expression in a mammary gland bioreactor [3,7,24,27]. Virus gene delivery or simple plasmid vector systems have been widely used for achieving transgenesis in cattle [8,11].…”

Molecular-based environmental risk assessment of three varieties of genetically engineered cows

“…a 1-15: transgenic-positive cows, 16: negative control cow produced by cross breeding between hLZ transgenic-positive F0 cow and non-transgenic bull, it was half-sibling of hLZ transgenic-positive F1 cow (4 or 5), 17-19: negative control cows produced by cross breeding between hLF transgenic-positive F0 cow and non-transgenic bull, they were half-sibling of hLF transgenic-positive F1 cow (8 or 9), 20: negative control cow produced by cross breeding between hLA transgenic-positive F0 cow and non-transgenic bull, it was half-sibling of hLA transgenic-positive F1 cow (13, 14, or 15), 21-23 negative control cow produced by cross breeding between non-transgenic cow and non-transgenic bull, and lived in the same barn as transgenic cows, 24-26: negative control cow produced by cross breeding between non-transgenic cow and non-transgenic bull, and lived in a different farm from transgenic cows b hLZ human lysozyme, hLF human lactoferrin, hLA human alpha lactalbumin c our previous study provided the detailed sequence information (Yu et al 2006;Liu et al 2008;Yang et al 2008;Wang et al 2008) d SCNT somatic cell nuclear transplantation Samples were then transferred to the laboratory and maintained at -70°C before use. Samples collected on 10 October 2009, 10 January 2010, 10 April 2010, and 10 July 2010 were designated as the autumn, winter, spring, and summer samples, respectively.…”