The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in Arabidopsis thaliana plants.
Here, we present the application of microbiology and biotechnology for the production of recombinant pharmaceutical proteins in plant cells. To the best of our knowledge and belief it is one of few examples of the expression of the prokaryotic staphylokinase (SAK) in the eukaryotic system. Despite the tremendous progress made in the plant biotechnology, most of the heterologous proteins still accumulate to low concentrations in plant tissues. Therefore, the composition of expression cassettes to assure economically feasible level of protein production in plants remains crucial. The aim of our research was obtaining a high concentration of the bacterial anticoagulant factor-staphylokinase, in Arabidopsis thaliana seeds. The coding sequence of staphylokinase was placed under control of the β-phaseolin promoter and cloned between the signal sequence of the seed storage protein 2S2 and the carboxy-terminal KDEL signal sequence. The engineered binary vector pATAG-sak was introduced into Arabidopsis thaliana plants via Agrobacterium tumefaciens-mediated transformation. Analysis of the subsequent generations of Arabidopsis seeds revealed both presence of the sak and nptII transgenes, and the SAK protein. Moreover, a plasminogen activator activity of staphylokinase was observed in the protein extracts from seeds, while such a reaction was not observed in the leaf extracts showing seed-specific activity of the β-phaseolin promoter.
Department of Genetics, Plant Molecular Biology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237, Lodz, Poland. kath@biol.uni.lodz.pl.
Katarzyna
K
Hnatuszko-Konka
Department of Genetics, Plant Molecular Biology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237, Lodz, Poland.
Piotr
P
Łuchniak
Department for Good Laboratory Practice, Bureau for Chemical Substances, Dowborczykow 30/34, 90-019, Lodz, Poland.
Aneta
A
Wiktorek-Smagur
Department of Genetics, Plant Molecular Biology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237, Lodz, Poland.
Aneta
A
Gerszberg
Department of Genetics, Plant Molecular Biology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237, Lodz, Poland.
Tomasz
T
Kowalczyk
Department of Immunoparasitology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237, Lodz, Poland.
Justyna
J
Gatkowska
Department of Genetics, Plant Molecular Biology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237, Lodz, Poland.
Andrzej K
AK
Kononowicz
D016960:Agrobacterium tumefaciens / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D003067:Coenzymes; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D005822:Genetic Vectors; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D058634:Molecular Farming / Q000379:methods*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
D003067:Coenzymes; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; C036483:T-DNA; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus
Agrobacterium; Molecular pharming; Plant expression cassette; Staphylokinase