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

Plant Biotechnol J. 2010 Feb;8(2):142-54. doi: 10.1111/j.1467-7652.2009.00470.x. Epub 2009 Dec 28.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cytosol / metabolism
  • Electrophoresis, Gel, Two-Dimensional
  • Gene Expression Regulation, Plant
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / metabolism
  • Protease Inhibitors / metabolism*
  • Proteome / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Solanum lycopersicum / genetics
  • Solanum tuberosum / genetics
  • Solanum tuberosum / metabolism*
  • Transgenes
  • alpha 1-Antichymotrypsin / genetics
  • alpha 1-Antichymotrypsin / metabolism

Substances

  • Plant Proteins
  • Protease Inhibitors
  • Proteome
  • Recombinant Proteins
  • alpha 1-Antichymotrypsin