Recombinant bovine alpha-lactalbumin obtained by limited proteolysis of a fusion protein expressed at high levels in Escherichia coli.
A cDNA clone containing the entire coding region for bovine pre-alpha-lactalbumin (LA) together with 27 base pairs of 5'-noncoding and 268 base pairs of 3'-noncoding sequences was isolated from a bovine mammary cDNA plasmid library in the Okayama-Berg vector system using a synthetic oligonucleotide probe and sequenced. The coding segment for mature LA was subcloned into the T7 expression system of Studier and co-workers (Studier, F.W., and Moffatt, B.A. (1986) J. Mol. Biol. 189, 113-130; Rosenberg, A.H., Lade, B.N., Chui, D.S., Lin, S.W., Dunn, J.J., and Studier, F.W. (1987) Gene (Amst.) 56, 125-135) and expressed as a 21-kDa fusion protein that consisted of the mature bovine LA sequence connected to the NH2-terminal 50 residues of human cathepsin D by a linker sequence containing protease cleavage sites. This fusion protein was expressed in an insoluble form and accumulated to about 50% of the total bacterial protein within 3 h after induction of T7 RNA polymerase synthesis. The protein was solubilized, purified by gel filtration, and converted to an active form by treatment with mixtures of reduced and oxidized glutathione in the presence of Ca2+. The maximum specific activity of the fusion protein was about 25% of that of native LA, suggesting that the attachment of an NH2-terminal extension sterically hinders but does not prevent the interaction with galactosyltransferase. The extension also does not block the binding of the regulatory Ca2+ ion that is required for folding from the reduced denatured state. Trypsin cleaved the folded fusion protein specifically at a Lys-Glu bond at the junction with the mature LA sequence to give a product indistinguishable in structure and activity from native LA.
Wang
M
M
Department of Biochemistry, University of Miami School of Medicine, Florida 33101.
Scott
W A
WA
Rao
K R
KR
Udey
J
J
Conner
G E
GE
Brew
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005796:Genes; D007768:Lactalbumin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008321:Mammary Glands, Animal / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D011498:Protein Precursors / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping
D015345:Oligonucleotide Probes; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C034435:pre-alpha-lactalbumin; D004247:DNA; D007768:Lactalbumin