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

Expression of recombinant human α-LA. (A) Schematic construct of phLa4-EGFP-NEO. The genomic human α-LA gene (9,459 bp) was inserted into the expression vector containing double selection marker gene EGFP and NPT II. The regions selected for PCR screening and Southern blot were indicated. P denoted product of PCR; P1 and P2 for human α-LA; and P3 and P4 for EGFP and NPT II, respectively. EGFP = enhanced green fluorescent protein; NPT II = neomycin phosphotransferase II; Amp = ampicillin-resistant gene; and ori = pBR322-derived origin. (B) PCR analysis of the transgenes. P1 and P2 represented the PCR products of recombinant human α-LA, respectively; P3 was for the PCR products of EGFP; and P4 for that of NPT II. (C) Southern blot analysis for transgenic human α-LA. The target DNA sequence was approximately 1.5kb, and approximately 1 copy of transgene was integrated into the genome of transgenic cows as a reference with copy numbers of positive control. (D) Western blot analysis for expression of recombinant human α-LA. The whey protein from transgenic and non-transgenic cows was separated on native PAGE and then blotted and hybridized with antihuman α-LA antibody. Lane 1 = human α-LA; lane 2 = human whey; lane 3 to 5 = transgenic whey from Xingwa, Longwa, and Huiwa, respectively; lane 6 = nontransgenic whey control.

Purification of recombinant human α-LA. (A) Anion exchange chromatography. The arrow indicates the fraction containing recombinant human α-LA free of bovine α-LA. (B) Gel filtration. The purified recombinant human α-LA was achieved by gel filtration chromatography. Purified recombinant protein was separated with native PAGE (upper insert in left panel) and then blotted for identification (lower insert in left panel). Human and bovine natural α-LA (hLA and bLA) were used as a positive and negative controls, respectively. The inset of gel electrophoresis showed the purity of recombinant protein separated by decreasing SDS-PAGE and stained by SYPRO Ruby gel stain (Invitrogen, Carlsbad, CA).

Lactose synthesis and Ca²⁺ binding shift. (A) Lactose synthesis stimulated by recombinant human α-LA in vitro. Human α-LA (hLA) was used as positive control. (B) Ca²⁺ binding shift on native PAGE. Human α-LA, bovine α-LA, and recombinant human α-LA were treated with CaCl₂ and ethylene glycol tetraacetic acid (EGTA), respectively. Conc = concentration.

Identification of glycosylation site of α-LA. (A) Electrospray ionization mass spectrometry for deglycosylated peptides. The circle indicated characteristic peptide for glycosylation, which was selected as a precursor ion for tandem mass spectrometry (MS/MS). (B) MS/MS for glycosylated peptide. Charge of fragment ions was +3, and Xcor was equal to 5.82. Glycosylation sequence and site (⁷¹Asn) are indicated on the top of this figure.

Analysis of milk composition. (A) Total fat, total protein, lactose, and total solids in transgenic and nontransgenic milk. (B) Total protein profile in skim milk from transgenic recombinant human α-LA (rhLA for lanes 4 to 6) and nontransgenic cows (bMilk for lanes 8 to 10) separated by decreasing SDS-PAGE. Lane 1 = molecular mass markers; lane 2 = the purified natural human α-LA (hLA); lane 3 = human skim milk (hMilk); lane 7 = transgenic cow skim milk containing recombinant human lactoferrin (rhLF). Ig_H = heavy chain of immunoglobin; LF = lactoferrin; Ig_L = light chain of immunoglobin; SA = serum albumin.

Analysis of protein composition by 2-dimensional electrophoresis. Transgenic and nontransgenic skim milk and whey samples were characterized at linear pH gradient 3 to 6 and 5 to 8. For comparison of skim milk, major proteins in the nontransgenic (A and B) and the transgenic (E and F) were separated. For comparison of proteins in whey, whey proteins in the nontransgenic (C and D) and the transgenic (G and H) were compared. LA = α-LA; LG A and B = A and B variants of β-LG; and Ig_H and Ig_L = heavy or light chains of immunoglobulin; hLA = human α-LA.