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

Abstract

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.

Beneficial ‘unintended effects’ of a cereal cystatin in transgenic lines of potato, Solanum tuberosum

“…In contrast with studies reporting significant effects for barley and strawberry cystatins [51,52], CCII had no significant effect on fungal biomass production (Student’s t -test; P =0.18) (Figure 5A). This observation, along with both the retention of CCII in the cytosol of potato leaf cells (see [21]) and the absence of Cys protease targets in the extracellular milieu (above), confirmed the relevance of B. cinerea as a pathogenic model to assess the impact of CCII-mediated, defense-related pleiotropic effects in the CCII-expressing lines, without the risk of confounding effects due to fungal protease inhibition.…”

“…Reverse transcriptase (RT) PCR, immunodetection and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI TOF MS) analyses were performed to select transgenic potato lines expressing CCII at different levels in leaves (Figure 1A,B), among a collection of independent transformants developed earlier in our laboratory [21]. CCII in these lines accumulates in the cytosolic compartment, under the control of the Cauliflower mosaic virus 35S constitutive promoter.…”

“…Kennebec (see ref. [21] for details on transgene construct). Line K used as parental line for genetic transformation was selected as a control for comparative purposes [9].…”

Functional proteomics-aided selection of protease inhibitors for herbivore insect control

“…Kennebec) expressing T6R or P2V were produced by Agrobacterium tumefaciens -mediated transformation of ‘line K’ axenic plantlets46 and selected in vitro using the neomycin phosphotransferase II selection marker for kanamycin resistance, as described earlier47. Gene constructs for transformation consisted of either cystatin-encoding sequences39 introduced between the NcoI and BsrGI cloning sites of a modified pUC19 vector (Fermentas Life Science) harbouring a duplicated version of the Cauliflower mosaic virus (CaMV) 35 S promoter, a tobacco etch virus enhancer sequence and the CaMV 35 S terminator sequence.…”

On-Chip Detection of Low-Molecular-Weight Recombinant Proteins in Plant Crude Extracts by SELDI-TOF MS

“…Several approaches are currently available to characterize the physicochemical and immunological properties of recombinant proteins (2) , including basic tools such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing and immunoblotting, and more sophisticated tools such as circular dichroism, nuclear magnetic resonance, capillary electrophoresis, electrospray ionization mass spectrometry (ESI MS), and matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS). As a complement, we recently discussed the potential of surface-enhanced laser desorption ionization time-of-flight MS ( SELDI-TOF MS ) for the rapid detection and quantitation of heterologous proteins in biological (plant) crude extracts (3) .…”