Cloning, expression, characterization and crystallization of BRP39, a signalling glycoprotein expressed during mammary gland apoptosis.
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 degrees 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 2.8A resolution. The crystals belonged to the space group C2 with unit cell parameters a=130.4A, b=81.3A, c=229.2A, beta=105.9 degrees. The structure refinement is in progress.
Mohanty
Ashok K
AK
Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India. ashokmohanty1@gmail.com
Fisher
Andrew J
AJ
Yu
Zhihao
Z
Pradeep
Mangottil A
MA
Janjanam
Jagdeesh
J
Kaushik
Jai K
JK
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D002686:Chitin / Q000378:metabolism; D000071451:Chitinase-3-Like Protein 1; D003001:Cloning, Molecular; D003460:Crystallization; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005260:Female; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment
C509289:Chil1 protein, mouse; D000071451:Chitinase-3-Like Protein 1; D018076:DNA, Complementary; D006023:Glycoproteins; D011994:Recombinant Proteins; D002686:Chitin; D018698:Glutamic Acid; D007930:Leucine