The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in Arabidopsis thaliana plants

Abstract

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.

High efficiency transformation of Brassica oleracea var. botrytis plants by Rhizobium rhizogenes

“…Therefore it is more likely that the heterologous protein is synthesised and gains its mature biological activity, and the given state of affairs may result from the extraction difficulties. Similar issues are widely described in the literature (Hnatuszko-Konka et al 2016). In addition, as it was tested by Barański and Puddephat (2004), in the cauliflower leaves glucuronidase expressions from the extensin promoter is among the lowest when compared to other organs.…”

Making plants into cost-effective bioreactors for highly active antimicrobial peptides

“…Alternatively, the AMPs might be expressed in transgenic grain seed, which tends to have yields higher than seen in leaf tissue. Recombinant proteins remain stable using traditional seed storage technique (Boothe et al, 2010;Morandini et al, 2011;Hnatuszko-Konka et al, 2016). Anionic AMPs used in the seed platform would address the increasing concern over the amount of antibiotics used with livestock (Ferber, 2003;Massé et al 2014;Hao et al, 2015).…”

Compendium on Food Crop Plants as a Platform for Pharmaceutical Protein Production

“…At this stage of cassette design, a decision on the selection and monitoring of transformant must be made as well. Therefore, selection or reporter genes (e.g., gus , gfp , kan ) should be present within the expression construct [ 34 , 35 , 36 ].…”

“…Leaf blades of ex vitro growing plants are most often infiltrated using a syringe, spraying method (agrospray), or vacuum infiltration. Additionally, in order to enhance transformation efficiency, the infiltration medium is enriched with surfactants [ 35 , 45 ].…”

“…In the next step, the obtained mixture is purified. The most commonly used extraction method from plant tissues is CTAB or SDS-based buffers [ 35 , 59 ]. Commercially available kits such as Qiagen DNeasy Mericon Kit or Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) can also be used [ 59 ].…”