Production of Desmodus rotundus salivary plasminogen activator α1 (DSPAα1) in tobacco is hampered by proteolysis

Abstract

The high fibrin specificity of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAalpha1 or desmoteplase (INN)) makes it a promising candidate for the treatment of acute ischemic stroke. In the current study we explored the use of transgenic tobacco plants and BY-2 suspension cells as alternative production platforms for this drug. Four different N-terminal signal peptides, from plants and animals, were used to translocate the recombinant DSPAalpha1 protein to the endomembrane system. Intact recombinant DSPAalpha1 was produced in transgenic plants and BY-2 cells, although a certain degree of degradation was observed in immunoblotted extracts. The choice of signal peptide had no major influence on the degradation pattern or recombinant protein accumulation, which reached a maximum level of 38 microg/g leaf material. N-terminal sequencing of purified, His6-tagged DSPAalpha1 revealed only minor changes in the position of signal peptide cleavage compared to the same protein expressed in Chinese hamster ovary cells. However, correctly processed recombinant DSPAalpha1 was also detected. The enzymatic activity of the recombinant protein was confirmed using an in vitro assay with unpurified and purified samples, demonstrating that plants are suitable for the production of functional DSPAalpha1. In contrast to whole plant cell extracts, no recombinant DSPAalpha1 was detected in the culture supernatant of transgenic BY-2 cells. Further analysis showed that recombinant DSPAalpha1 is subject to proteolysis and that endogenous secreted BY-2 proteases are responsible for DSPAalpha1 degradation in the culture medium. The addition of a highly concentrated protease inhibitor mixture or 5 mM EDTA reduced DSPAalpha1 proteolysis, improving the accumulation of intact product in the culture medium. Strategies to improve the plant cell suspension system for the production of secreted recombinant proteins are discussed.

Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells

“…The production of recombinant DSPAa1 in BY-2 cells is hampered by the activity of a metalloproteinase that can be inhibited to a certain extent by the addition of EDTA (Schiermeyer et al 2005). To determine whether NtMMP1 is a candidate for this metalloproteinase, NtMMP1 and DSPAa1 were incubated together and residual DSPAa1 activity was determined using the specific substrate S2288.…”

“…Proteolysis can be particularly challenging when recombinant proteins are targeted for secretion to the apoplast or into the culture medium (Delannoy et al 2008). For example, plasminogen activator DSPAa1 from the vampire bat Desmodus rotundus is almost completely degraded by secreted proteases when expressed in tobacco Bright Yellow 2 (BY-2) suspension cells (Schiermeyer et al 2005). However, the yield of recombinant DSPAa1 is improved by the addition of the metalloprotease inhibitor EDTA, suggesting that metalloproteases are the predominant source of secreted protease activity (Schiermeyer et al 2005).…”

“…For example, plasminogen activator DSPAa1 from the vampire bat Desmodus rotundus is almost completely degraded by secreted proteases when expressed in tobacco Bright Yellow 2 (BY-2) suspension cells (Schiermeyer et al 2005). However, the yield of recombinant DSPAa1 is improved by the addition of the metalloprotease inhibitor EDTA, suggesting that metalloproteases are the predominant source of secreted protease activity (Schiermeyer et al 2005). It is well known that the proteolytic degradation of some heterologous proteins can be reduced by additives such as PVP or gelatin (LaCount et al 1997;Lee et al 2002) but the inhibitory effect is seldom complete and the protease inhibitors can adversely affect cell growth (Schiermeyer et al 2005).…”

Transgenic Plants as Biofactories for the Production of Biopharmaceuticals: A Case Study of Human Placental Lactogen

“…In eukaryotes this process is thought to follow the scanning mechanism conducted by small subunit of ribosome from 5'cap of mRNA through the untranslated leader until the first start codon (AUG) is found (Kermode, 2006). Including 5' UTR sequences from Alfalfa mosaic virus (AMV), Tobacco mosaic virus (TMV), Chalcone synthase (CHS), or Alcohol dehydrogenase (NtADH) have been successfully used to enhance the translation efficiency, allowing transgene levels 30 to 100-fold higher (Satoh et al, 2004;Schiermeyer et al, 2005).…”

Plant Bioreactors for Pharmaceuticals

“…For example, Bacitracin was applied to the suspension culture of transgenic tobacco (Nicotiana tobacum) cells, resulting in a 2.18-fold improvement in yield of recombinant human granulocytemacrophage colony-stimulating factor (hGM-CSF) (Lee et al, 2003). The addition of PIs in transgenic tobacco BY-2 suspension culture also reduced the proteolysis of the recombinant Desmodus rotundus salivary plasminogen activator alpha1, improving the accumulation of intact product (Schiermeyer et al, 2005). Although natural or synthetic exogenous PIs could provide protection against the extracellular proteases, the use of these additives results in an increased cost due to the need of additional purification steps to remove them.…”