A companion protease inhibitor for the protection of cytosol-targeted recombinant proteins in plants.
We reported earlier the potential of tomato cathepsin D inhibitor (SlCDI) as an in-built 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 SlCDI for the in situ protection of proteins in potato leaves. Total protein assays with control and SlCDI-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 approximately 700 proteins detected on 2-D gels, only 20 exhibited a significantly altered level on a protein-specific basis, whereas most proteins were up-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 SlCDI-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 SlCDI-expressing lines, suggest a general, proteasome-independent protein stabilizing effect of SlCDI in planta. Transient expression assays with human alpha(1)-antichymotrypsin also showed a stabilizing effect for SlCDI on heterologous proteins, leading to net levels of the human protein increased by approximately 2.5-fold in SlCDI-expressing plants. These data illustrate, overall, the potential of SlCDI as an in vivo protein-stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation.
Goulet
Charles
C
CRH/INAF, Pavillon des Services (INAF), Université Laval, Québec, QC, Canada.
Benchabane
Meriem
M
Anguenot
Raphaël
R
Brunelle
France
F
Khalf
Moustafa
M
Michaud
Dominique
D
D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D018506:Gene Expression Regulation, Plant; D018551:Solanum lycopersicum / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D011198:Solanum tuberosum / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes; D000514:alpha 1-Antichymotrypsin / Q000235:genetics / Q000378:metabolism
D010940:Plant Proteins; D011480:Protease Inhibitors; D020543:Proteome; D011994:Recombinant Proteins; D000514:alpha 1-Antichymotrypsin