Translocation of aprotinin, a therapeutic protease inhibitor, into the thylakoid lumen of genetically engineered tobacco chloroplasts

Abstract

SummaryAprotinin, a bovine protease inhibitor of important therapeutic value, was expressed in tobacco plastid transformants. This disulphide bond-containing protein was targeted to the lumen of thylakoids using signal peptides derived from nuclear genes which encode lumenal proteins. Translocation was attempted via either the general secretion (Sec) or the twin-arginine translocation (Tat) pathway. In both cases, this strategy allowed the production of genuine aprotinin with its N-terminal arginine residue. The recombinant protease inhibitor was efficiently secreted within the lumen of thylakoids, accumulated in older leaves and was bound to trypsin, suggesting that the three disulphide bonds of aprotinin are correctly folded and paired in this chloroplast compartment. Mass spectrometric analysis indicated that translocation via the Sec pathway, unlike the Tat pathway, led predominantly to an oxidized protein. Translocation via the Tat pathway was linked to a slightly decreased growth rate, a pale-green leaf phenotype and supplementary expression products associated with the thylakoids.

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

“…Aprotinin, a bovine protease inhibitor, was targeted to the lumen of thylakoids of tobacco plastid transformants using thylakoid signal peptides derived from nuclear genes which encode luminal proteins (Tissot et al 2008). Plastid-encoded pre-Tic40 was properly processed and targeted and assembled into a functional complex at the inner membrane of chloroplast (Singh et al 2008).…”

Plant Physiological Adaptations to the Massive Foreign Protein Synthesis Occurring in Recombinant Chloroplasts

“…In most reports, which include an insecticidal toxin expressed at 46% tsp (De Cosa et al, 2001), or a GFP expressed at 38% tsp (Yabuta et al, 2008), no obvious phenotypic defect, such as growth retardation, has been observed in plastid transformants. When phenotypic modifications were noted, these were directly linked to the specific properties of the expressed transgenes (Tregoning et al, 2003;Magee et al, 2004;Ruiz and Daniell, 2005;Chakrabarti et al, 2006;Hasunuma et al, 2008;Tissot et al, 2008). Only in the case of lysin was the hyperexpression of the recombinant protein reported to limit plant development by exhausting the protein synthetic capacity of chloroplasts (Oey et al, 2009).…”

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

“…The transplastomic plant lines with the transgene expression cassette controlled by the strong constitutive ribosomal RNA operon promoter showed distinct growth retardations and mild pigment deficiency. In many cases, plastid expression of recombinant proteins does not result in abnormal phenotypes, but an increasing number of studies has reported phenotypic alterations in transplastomic plants (Lössl et al 2003;Magee et al 2004;Scotti et al 2015;Tissot et al 2008;Tregoning et al 2003;Waheed et al 2011). The two main reasons underlying pigment deficiency or a delay in plant development are toxicity of the transgene product due to interference of the recombinant protein with essential processes in the chloroplast (e.g.…”