A companion protease inhibitor for the protection of cytosol-targeted recombinant proteins in plants

We reported earlier the potential of tomato cathepsin D inhibitor (S/CDI) as an inbuilt stabilizing agent for the protection of recombinant proteins in transgenic plant leaf crude extracts (Plant Biotechnol J 4, 359-368) Here we document the potential of S/CDI for the in situ protection of proteins in potato leaves Total protein assays with control and SICIDI-expressing potato lines indicated a Positive impact of slcdi transgene expression on leaf protein content, with a mean relative increase of 35%-40% depending on the light regime Out of -700 proteins detected on 2-D gels, only 20 exhibited a significantly altered level on a protein-specific basis, whereas most proteins were u.p-regulated on a leaf fresh weight basis, albeit at variable rates Quantitative reverse trancriptase-PCR assays for rubisco activase showed similar transcript levels in leaves of test and control lines despite protein levels increased by two- to threefold in SICDI-expressing lines These observations, along with the unrelated biological functions assigned to MS-identified proteins up-regulated in leaves and protease assays showing slightly increased proteasome activity in protein extracts of S/CDI-expressing lines, suggest a general, proteasome-independent protein stabilizing effect of SICD1 in planta Transient expression assays with human alpha 1-antichymotrypsin also showed a stabilizing effect for S/CDl on heterologous proteins, leading to net levels of the human protein increased by -2.5-fold in SIC D I -expressing plants These data illustrate, overall, the potential of S/CDl as an In vivo protein-stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation