Companion protease inhibitors to protect recombinant proteins in transgenic plant extracts.

We describe a general approach for the use of recombinant protease inhibitors as stabilizing agents for clinically useful proteins extracted from transgenic plant tissues. A procedure is first described to assess the overall (in)stability of heterologous proteins in transgenic plant crude protein extracts. Step-by-step protocols are then presented for the choice and use of companion protease inhibitors inhibiting the host plant proteases during extraction.

Unintended molecular interactions in transgenic plants expressing clinically useful proteins: the case of bovine aprotinin traveling the potato leaf cell secretory pathway.

We assessed the impact of subcellular targeting on the heterologous expression of a clinically useful protease inhibitor, bovine aprotinin, in leaves of potato, Solanum tuberosum. Transgenic potato lines targeting aprotinin to the cytosol, the ER or the apoplast were first generated, and then assessed for their ability to accumulate the recombinant protein. On-chip detection and quantitation of aprotinin variants by SELDI TOF MS showed the inhibitor to be absent in the cytosol, but present under different forms in the ER and the apoplast.

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.

Preventing unintended proteolysis in plant protein biofactories.

Numerous reports have been published over the last decade assessing the potential of plants as useful hosts for the heterologous expression of clinically useful proteins. Significant progress has been made, in particular, in optimizing transgene transcription and translation in plants, and in elucidating the complex post-translational modifications of proteins typical of the plant cell machinery. In this article, we address the important issue of recombinant protein degradation in plant expression platforms, which directly impacts on the final yield, homogeneity and overall quality of the resulting protein product.

MsCYS1, a developmentally-regulated cystatin from alfalfa.

Several roles have been attributed to cystatins in plants, ranging from the regulation of host [endogenous] cysteine proteases to the inhibition of herbivorous pest [exogenous] proteases. We report here the cloning, expression and functional characterization of a novel cystatin from alfalfa, Medicago sativa L. The new sequence, isolated from a cDNA expression library prepared from young leaves, encodes a protein, MsCYS1, with the typical inhibitory motifs of cystatins, namely the central signature motif QxVxG, a GG doublet in the N-terminal trunk, and a W residue in the C-terminal region, about 30 amino acids distant from the central inhibitory motif.

A multicomponent, elicitor-inducible cystatin complex in tomato, Solanum lycopersicum.

We assessed the ability of the fungal elicitor arachidonic acid to induce cystatin genes in tomato (Solanum lycopersicum), using a cDNA expression library from arachidonate-treated leaves. The cDNAs of two novel cystatins were isolated, coding for an approx. 11-kDa protein, SlCYS10; and for a 23.

An in-built proteinase inhibitor system for the protection of recombinant proteins recovered from transgenic plants.

Proteolytic degradation represents a significant barrier to the efficient production of several recombinant proteins in plants, both in vivo during their expression and in vitro during their recovery from source tissues. Here, we describe a strategy to protect recombinant proteins during the recovery process, based on the coexpression of a heterologous proteinase inhibitor acting as a 'mouse trap' against the host proteases during extraction. After confirming the importance of trypsin- and chymotrypsin-like activities in crude protein extracts of potato (Solanum tuberosum L.

Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites.

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots).

Colorado potato beetles show differential digestive compensatory responses to host plants expressing distinct sets of defense proteins.

Herbivorous insects fed plants expressing proteinase inhibitors (PIs) compensate for the loss of digestive proteolytic functions by producing novel proteinases. We assessed here whether such compensatory responses represent a general, non-specific adaptation to defense-related proteins in host plant tissues, or if distinct responses occur depending on the stress exerted on the plant. As a model, growth, development, and digestive proteases of the Colorado potato beetle (Leptinotarsa decemlineata Say) were monitored after feeding larvae with plants pre-treated with either methyl jasmonate or arachidonic acid, two compounds inducing different sets of defense genes in potato.