A SELDI-TOF MS procedure for the detection, quantitation, and preliminary characterization of low-molecular-weight recombinant proteins expressed in transgenic plants

被引：14

作者：

Badri, M. Amine ^{[1
]}

Rivard, Daniel ^{[1
]}

Coenen, Karine ^{[1
]}

Vaillancourt, Louis-Philippe ^{[1
]}

Goulet, Charles ^{[1
]}

Michaud, Dominique ^{[1
]}

机构：

[1] Univ Laval, INAF, Pavillon Serv, Dept Phytol,CRH, Quebec City, PQ G1V 0A6, Canada

来源：

PROTEOMICS | 2009年 / 9卷 / 02期

基金：

加拿大自然科学与工程研究理事会;

关键词：

Bovine aprotinin; Corn cystatin II; Recombinant protein quantitation; Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS); Transgenic plants; THERAPEUTIC PROTEINS; INHIBITOR; SYSTEMS; IDENTIFICATION; TECHNOLOGY; PRODUCTS; TOOL;

D O I：

10.1002/pmic.200700233

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

We describe a SELDI-TOF MS procedure for the rapid detection and quantitation of low-molecular-weight recombinant proteins expressed in plant. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens-mediated transformation, and then used as test material for the analyses. Real-time RT-PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI-TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure,carried out in less than 2 h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity, and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production.

引用

页码：233 / 241

页数：9

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