title: Expression of recombinant staphylokinase, a fibrin-specific plasminogen activator of bacterial origin, in potato (Solanum tuberosum L.) plants.
abstract: One of the most dynamically developing sectors of green biotechnology is molecular farming using transgenic plants as natural bioreactors for the large scale production of recombinant proteins with biopharmaceutical and therapeutic values. Such properties are characteristic of certain proteins of bacterial origin, including staphylokinase. For many years, work has been carried out on the use of this protein in thrombolytic therapy. In this study, transgenic Solanum tuberosum plants expressing a CaMV::sak-mgpf-gusA gene fusion, were obtained. AGL1 A. tumefaciens strain was used in the process of transformation. The presence of the staphylokinase gene was confirmed by PCR in 22.5% of the investigated plants. The expression of the fusion transgene was detected using the β-glucuronidase activity assay in 32 putative transgenic plants. Furthermore, on the basis of the GUS histochemical reaction, the transgene expression pattern had a strong, constitutive character in seven of the transformants. The polyacrylamide gel electrophoresis of a protein extract from the SAK/PCR-positive plants, revealed the presence of a119 kDa protein that corresponds to that of the fusion protein SAK-mGFP-GUSA. Western blot analysis, using an antibody against staphylokinase, showed the presence of the staphylokinase domain in the 119 kDa protein in six analyzed transformants. However, the enzymatic test revealed amidolytic activity characteristic of staphylokinase in the protein extract of only one plant. This is the first report on a Solanum tuberosum plant producing a recombinant staphylokinase protein, a plasminogen activator of bacterial origin.
authors.affiliation: Department of Genetics and Plant Molecular Biology and Biotechnology, The University of Lodz, Banacha Street 12/16, 90-237 Lodz, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Gerszberg
authors.forename: Aneta
authors.initials: A
authors.lastname: Wiktorek-Smagur
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Hnatuszko-Konka
authors.forename: Piotr
authors.initials: P
authors.lastname: Łuchniak
authors.forename: Andrzej K
authors.initials: AK
authors.lastname: Kononowicz
mesh_terms: D001709:Biotechnology / Q000379:methods; D015153:Blotting, Western; D017796:Caulimovirus / Q000235:genetics; D020869:Gene Expression Profiling; D005822:Genetic Vectors; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics; D008970:Molecular Weight; D030821:Plants, Genetically Modified; D010960:Plasminogen Activators / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011198:Solanum tuberosum / Q000235:genetics*; D013678:Technology, Pharmaceutical / Q000379:methods
chemical_list: D011993:Recombinant Fusion Proteins; D010960:Plasminogen Activators; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus