Cloning, expression, characterization and crystallization of BRP39, a signalling glycoprotein expressed during mammary gland apoptosis
Cloning, expression, characterization and crystallization of BRP39, a signalling glycoprotein expressed during mammary gland apoptosis
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DOI：
10.1016/j.pep.2008.11.001
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发表时间：
2009-04-01
影响因子：
1.6
通讯作者：
Kaushik, Jai K.
中科院分区：
文献类型：
--
作者：
Mohanty, Ashok K.;Fisher, Andrew J.;Kaushik, Jai K.
乳腺退化蛋白（BRP39）是一种糖蛋白，在小鼠乳腺退化过程中表达。BRP39的生理功能尚不清楚。BRP39的高水平表达也与乳腺癌的发展有关。在本研究中，通过PCR技术克隆了编码rBRP39（重组BRP39）的cDNA。它由1，143个核苷酸组成，编码一个381个氨基酸残基的开放阅读框架，包括21个氨基酸的信号序列。重组BRP39在大肠杆菌中以可溶形式在低温（15 ° C）下产生。通过Western blot分析证实rBRP39的表达和纯化。纯化的rBRP39具有较高的几丁质结合活性，但没有几丁质酶活性。BRP39几丁质酶活性的缺失可能与其关键活性位点Glu120突变为Leu120和Asp118突变为Ala118有关。然而，通过定点诱变将残基回复为Glu的突变体不显示几丁质酶活性。使纯化的重组BRP39结晶，晶体衍射X射线至2.8埃分辨率。晶体属于空间群C2，晶胞参数a = 130.4埃，B = 81.3埃，c = 229.2埃，β = 105.9度。结构优化正在进行中。(C)2008年爱思唯尔公司All rights reserved.
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 Glul 20 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 angstrom resolution. The crystals belonged to the space group C2 with unit cell parameters a = 130.4 angstrom, b = 81.3 angstrom, c = 229.2 angstrom, beta = 105.9 degrees. The structure refinement is in progress. (C) 2008 Elsevier Inc. All rights reserved.