Translocation of aprotinin, a therapeutic protease inhibitor, into the thylakoid lumen of genetically engineered tobacco chloroplasts.
Aprotinin, 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.
Tissot
Ghislaine
G
Bayer BioScience, 69263 Lyon Cedex 09, France.
Canard
Hélène
H
Nadai
Marie
M
Martone
Aris
A
Botterman
Johan
J
Dubald
Manuel
M
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D005818:Genetic Engineering; D018515:Plant Leaves / Q000166:cytology; D030821:Plants, Genetically Modified; D011480:Protease Inhibitors / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins; D020524:Thylakoids; D014026:Nicotiana / Q000166:cytology* / Q000235:genetics*
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D007611:Aprotinin