Cloning, expression, characterization and crystallization of BRP39, a signalling glycoprotein expressed during mammary gland apoptosis

Ashok K. Mohanty, Andrew J Fisher, Zhihao Yu, Mangottil A. Pradeep, Jagdeesh Janjanam, Jai K. Kaushik

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Breast regression protein (BRP39) is a glycoprotein, which is expressed during mammary gland involution in mouse. The physiological function of BRP39 is not known. High levels of expression of BRP39 have also been associated with breast cancer development. In the present investigation a cDNA encoding rBRP39 (recombinant BRP39) was cloned by PCR techniques. It consists of 1,143 nucleotides and encodes an open reading frame of 381 amino acid residues including a signal sequence of 21 amino acids. Recombinant BRP39 was produced in E. coli in a soluble form at low temperature (15 °C). Expression and purification of rBRP39 was confirmed by western blot analysis. Purified rBRP39 showed high chitin-binding activity but no chitinase activity. The lack of chitinase activity may be attributed to the mutation of critical active site residue Glu120 to Leu120 and Asp118 to Ala118 in BRP39. However, a mutant in which the residue was reverted back to Glu, by site directed mutagenesis, displayed no chitinase activity. Purified recombinant BRP39 was crystallized and the crystals diffracted X-rays to 2.8 Å resolution. The crystals belonged to the space group C2 with unit cell parameters a = 130.4 Å, b = 81.3 Å, c = 229.2 Å, β = 105.9°. The structure refinement is in progress.

Original languageEnglish (US)
Pages (from-to)213-218
Number of pages6
JournalProtein Expression and Purification
Volume64
Issue number2
DOIs
StatePublished - Apr 2009

Fingerprint

Chitinases
Human Mammary Glands
Crystallization
Organism Cloning
Glycoproteins
Apoptosis
Amino Acids
Chitin
Protein Sorting Signals
Site-Directed Mutagenesis
Open Reading Frames
Catalytic Domain
Breast
Nucleotides
Complementary DNA
Western Blotting
X-Rays
Breast Neoplasms
Escherichia coli
Polymerase Chain Reaction

Keywords

  • BRP39
  • Chitinase-like protein
  • Crystallization
  • Involution

ASJC Scopus subject areas

  • Biotechnology

Cite this

Cloning, expression, characterization and crystallization of BRP39, a signalling glycoprotein expressed during mammary gland apoptosis. / Mohanty, Ashok K.; Fisher, Andrew J; Yu, Zhihao; Pradeep, Mangottil A.; Janjanam, Jagdeesh; Kaushik, Jai K.

In: Protein Expression and Purification, Vol. 64, No. 2, 04.2009, p. 213-218.

Research output: Contribution to journalArticle

Mohanty, Ashok K. ; Fisher, Andrew J ; Yu, Zhihao ; Pradeep, Mangottil A. ; Janjanam, Jagdeesh ; Kaushik, Jai K. / Cloning, expression, characterization and crystallization of BRP39, a signalling glycoprotein expressed during mammary gland apoptosis. In: Protein Expression and Purification. 2009 ; Vol. 64, No. 2. pp. 213-218.
@article{b71ee5bf0fec40bbbc85a630f843b30a,
title = "Cloning, expression, characterization and crystallization of BRP39, a signalling glycoprotein expressed during mammary gland apoptosis",
abstract = "Breast regression protein (BRP39) is a glycoprotein, which is expressed during mammary gland involution in mouse. The physiological function of BRP39 is not known. High levels of expression of BRP39 have also been associated with breast cancer development. In the present investigation a cDNA encoding rBRP39 (recombinant BRP39) was cloned by PCR techniques. It consists of 1,143 nucleotides and encodes an open reading frame of 381 amino acid residues including a signal sequence of 21 amino acids. Recombinant BRP39 was produced in E. coli in a soluble form at low temperature (15 °C). Expression and purification of rBRP39 was confirmed by western blot analysis. Purified rBRP39 showed high chitin-binding activity but no chitinase activity. The lack of chitinase activity may be attributed to the mutation of critical active site residue Glu120 to Leu120 and Asp118 to Ala118 in BRP39. However, a mutant in which the residue was reverted back to Glu, by site directed mutagenesis, displayed no chitinase activity. Purified recombinant BRP39 was crystallized and the crystals diffracted X-rays to 2.8 {\AA} resolution. The crystals belonged to the space group C2 with unit cell parameters a = 130.4 {\AA}, b = 81.3 {\AA}, c = 229.2 {\AA}, β = 105.9°. The structure refinement is in progress.",
keywords = "BRP39, Chitinase-like protein, Crystallization, Involution",
author = "Mohanty, {Ashok K.} and Fisher, {Andrew J} and Zhihao Yu and Pradeep, {Mangottil A.} and Jagdeesh Janjanam and Kaushik, {Jai K.}",
year = "2009",
month = "4",
doi = "10.1016/j.pep.2008.11.001",
language = "English (US)",
volume = "64",
pages = "213--218",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Cloning, expression, characterization and crystallization of BRP39, a signalling glycoprotein expressed during mammary gland apoptosis

AU - Mohanty, Ashok K.

AU - Fisher, Andrew J

AU - Yu, Zhihao

AU - Pradeep, Mangottil A.

AU - Janjanam, Jagdeesh

AU - Kaushik, Jai K.

PY - 2009/4

Y1 - 2009/4

N2 - Breast regression protein (BRP39) is a glycoprotein, which is expressed during mammary gland involution in mouse. The physiological function of BRP39 is not known. High levels of expression of BRP39 have also been associated with breast cancer development. In the present investigation a cDNA encoding rBRP39 (recombinant BRP39) was cloned by PCR techniques. It consists of 1,143 nucleotides and encodes an open reading frame of 381 amino acid residues including a signal sequence of 21 amino acids. Recombinant BRP39 was produced in E. coli in a soluble form at low temperature (15 °C). Expression and purification of rBRP39 was confirmed by western blot analysis. Purified rBRP39 showed high chitin-binding activity but no chitinase activity. The lack of chitinase activity may be attributed to the mutation of critical active site residue Glu120 to Leu120 and Asp118 to Ala118 in BRP39. However, a mutant in which the residue was reverted back to Glu, by site directed mutagenesis, displayed no chitinase activity. Purified recombinant BRP39 was crystallized and the crystals diffracted X-rays to 2.8 Å resolution. The crystals belonged to the space group C2 with unit cell parameters a = 130.4 Å, b = 81.3 Å, c = 229.2 Å, β = 105.9°. The structure refinement is in progress.

AB - Breast regression protein (BRP39) is a glycoprotein, which is expressed during mammary gland involution in mouse. The physiological function of BRP39 is not known. High levels of expression of BRP39 have also been associated with breast cancer development. In the present investigation a cDNA encoding rBRP39 (recombinant BRP39) was cloned by PCR techniques. It consists of 1,143 nucleotides and encodes an open reading frame of 381 amino acid residues including a signal sequence of 21 amino acids. Recombinant BRP39 was produced in E. coli in a soluble form at low temperature (15 °C). Expression and purification of rBRP39 was confirmed by western blot analysis. Purified rBRP39 showed high chitin-binding activity but no chitinase activity. The lack of chitinase activity may be attributed to the mutation of critical active site residue Glu120 to Leu120 and Asp118 to Ala118 in BRP39. However, a mutant in which the residue was reverted back to Glu, by site directed mutagenesis, displayed no chitinase activity. Purified recombinant BRP39 was crystallized and the crystals diffracted X-rays to 2.8 Å resolution. The crystals belonged to the space group C2 with unit cell parameters a = 130.4 Å, b = 81.3 Å, c = 229.2 Å, β = 105.9°. The structure refinement is in progress.

KW - BRP39

KW - Chitinase-like protein

KW - Crystallization

KW - Involution

UR - http://www.scopus.com/inward/record.url?scp=58749094953&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=58749094953&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2008.11.001

DO - 10.1016/j.pep.2008.11.001

M3 - Article

VL - 64

SP - 213

EP - 218

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 2

ER -