A SELDI-TOF MS procedure for the detection, quantitation, and preliminary characterization of low-molecular-weight recombinant proteins expressed in transgenic plants.
We describe a SELDI-TOF MS procedure for the rapid detection and quantitation of low-molecular-weight recombinant proteins expressed in plants. 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.
Badri
M Amine
MA
Département de phytologie, CRH/INAF, Université Laval, Québec, Canada.
Rivard
Daniel
D
Coenen
Karine
K
Vaillancourt
Louis-Philippe
LP
Goulet
Charles
C
Michaud
Dominique
D
D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D015891:Cystatins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D016018:Least-Squares Analysis; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D011198:Solanum tuberosum / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D015891:Cystatins; D010940:Plant Proteins; D011994:Recombinant Proteins; C077834:cystatin protein, Zea mays; D007611:Aprotinin