Preventing unintended proteolysis in plant protein biofactories

Abstract

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. Unlike several more stable and structurally less complex pharmaceuticals, recombinant proteins present a natural tendency to structural heterogeneity, resulting in part from the inherent instability of polypeptide chains expressed in heterologous environments. Proteolytic processing, notably, may dramatically alter the structural integrity and overall accumulation of recombinant proteins in plant expression systems, both in planta during expression and ex planta after extraction. In this article, we describe the current strategies proposed to minimize protein hydrolysis in plant protein factories, including organ-specific transgene expression, organelle-specific protein targeting, the grafting of stabilizing protein domains to labile proteins, protein secretion in natural fluids and the co-expression of companion protease inhibitors.

Figure 1

Subcellular targeting of recombinant proteins in plant cells. Recombinant proteins bearing an N‐terminal signal peptide (SP) in their primary sequence enter the cell secretory pathway via the endoplasmic reticulum (ER), and then travel through the Golgi system to be secreted in the apoplast (default pathway) or directed to the vacuole if a vacuolar sorting determinant (VSD) is present in the protein sequence. Proteins secreted into the ER can also be retained in this compartment by the grafting of an ER retention signal – the KDEL (or HDEL) tetrapeptide motif – at the C‐terminus. Proteins with no signal peptide accumulate in the cytosol (default location) or migrate to specific organelles when an appropriate peptide signal is included in the transgene sequence. Peptide signals used recently in transgenic plant platforms include plastid (e.g. chloroplast) transit peptides (PTP), nuclear localization signals (NLS) and the tripeptide peroxisome target sequence serine‐lysine‐leucine (SKL).

Figure 2

Stabilization of exogenous proteins in plant crude protein extracts by phenylmethylsulphonyl fluoride (PMSF) and chymostatin (CHY). The example is human fibronectin (hFb) challenged with alfalfa leaf proteases. hFb was first incubated for 10 min at 20 °C with major protease fractions of alfalfa leaf proteases (P1 or P2) in the presence or absence of 0.1 mm PMSF or 10 µm CHY, and then immunodetected using appropriate anti‐hFb polyclonal antibodies. CTL, control extract with no inhibitor added.