Recombinant bovine α-lactalbumin obtained by limited proteolysis of a fusion protein expressed at high levels in Escherichia coli

M. Wang, W. A. Scott, K. R. Rao, J. Udey, Gregory E Conner, K. Brew

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

A cDNA clone containing the entire coding region for bovine pre-α-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 of 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 function 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.

Original languageEnglish
Pages (from-to)21116-21121
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number35
StatePublished - Dec 1 1989

Fingerprint

Proteolysis
Lactalbumin
Escherichia coli
Fusion reactions
Proteins
Base Pairing
lysylglutamic acid
Complementary DNA
Galactosyltransferases
Cathepsin D
Bacterial Proteins
Glutathione Disulfide
Oligonucleotide Probes
Gene Library
Trypsin
Gel Chromatography
Glutathione
Breast
Plasmids
Peptide Hydrolases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Recombinant bovine α-lactalbumin obtained by limited proteolysis of a fusion protein expressed at high levels in Escherichia coli. / Wang, M.; Scott, W. A.; Rao, K. R.; Udey, J.; Conner, Gregory E; Brew, K.

In: Journal of Biological Chemistry, Vol. 264, No. 35, 01.12.1989, p. 21116-21121.

Research output: Contribution to journalArticle

@article{e694622088fd484994f737795aa6b919,
title = "Recombinant bovine α-lactalbumin obtained by limited proteolysis of a fusion protein expressed at high levels in Escherichia coli",
abstract = "A cDNA clone containing the entire coding region for bovine pre-α-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 of 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 function 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.",
author = "M. Wang and Scott, {W. A.} and Rao, {K. R.} and J. Udey and Conner, {Gregory E} and K. Brew",
year = "1989",
month = "12",
day = "1",
language = "English",
volume = "264",
pages = "21116--21121",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "35",

}

TY - JOUR

T1 - Recombinant bovine α-lactalbumin obtained by limited proteolysis of a fusion protein expressed at high levels in Escherichia coli

AU - Wang, M.

AU - Scott, W. A.

AU - Rao, K. R.

AU - Udey, J.

AU - Conner, Gregory E

AU - Brew, K.

PY - 1989/12/1

Y1 - 1989/12/1

N2 - A cDNA clone containing the entire coding region for bovine pre-α-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 of 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 function 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.

AB - A cDNA clone containing the entire coding region for bovine pre-α-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 of 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 function 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.

UR - http://www.scopus.com/inward/record.url?scp=0024834559&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024834559&partnerID=8YFLogxK

M3 - Article

VL - 264

SP - 21116

EP - 21121

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 35

ER -