Redesigning amino/carboxyl ends of DARPin G3 for high thermostability and production in tobacco transplastomic plants.

Redesigning the N- and C-capping repeats of the native DARPin G3 significantly improved its stability, and may facilitate its purification from the total soluble proteins of high-temperature dried leaf materials of transplastomic plants. Designed ankyrin repeat proteins (DARPins) constitute a promising class of binding molecules that can overcome the limitations of monoclonal antibodies and enable the development of novel therapeutic approaches. Despite their inherent stability, detailed studies have revealed that the original capping repeats derived from natural ankyrin repeat proteins impair the stability of the initial DARPin design.

The lyophilized chloroplasts store synthetic DARPin G3 as bioactive encapsulated organelles.

Background: The high cost of fermentation, purification, cold storage and transportation, short shelf life, and sterile delivery methods of biopharmaceuticals, is a matter for producers and consumers as well. Since the FDA has now approved plant cells for large-scale, cost-effective biopharmaceutical production, the isolation and lyophilization of transplastomic chloroplasts can cover concerns about limitations. DARPins are engineered small single-domain proteins that have been selected to bind to HER2 with high affinity and specificity.

The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology.

Background: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets.

Computer-Based Tools Unmask Critical Mineral Nutrient Interactions in Hoagland Solution for Healthy Kiwiberry Plant Acclimatization.

The aim of this study was to better understand the response of acclimatized plants grown to a set of mineral nutrient combinations based on Hoagland solution. To reach that, two computer-based tools were used: the design of experiments (DOE) and a hybrid artificial intelligence technology that combines artificial neural networks with fuzzy logic. DOE was employed to create a five-dimensional IV-design space by categorizing all macroelements and one microelement (copper) of Hoagland mineral solution, reducing the experimental design space from 243 (3) to 19 treatments.

Functional fusion expression of sunflower multicystatin in E. coli and its comparison with a single domain cystatin.

Identification of the molecular structure and novel biophysiological functions of plant cystatins or phytocystatins is of great interest in the field of molecular biology. The important requirements for these are the efficient production, purification and correctly folded forms of these proteins. We report here the cloning, easy expression and characterization of a sunflower multicystatin (SMC) as a functional fusion protein in E.

Carborundum-dependent entrance of EcoRI restriction enzyme into plant cells and specific cleavage of genomic DNA.

In a basic research to determine the morpho-molecular interactions of plant tissues with EcoRI DNA restriction enzyme, it was demonstrated that this protein is capable of entering the sunflower and maize leaf cells using a plant tissue-abrading material and cleaving the genomic DNA at specific sites. This was inferred from the analysis of morphological patterns of EcoRI-treated leaf areas as well as using some molecular tests, including the cleavage pattern analysis of genomic DNA isolated from treated locations followed by ligation of cleaved fragments into EcoRI site of a DNA cloning vector system. The overall results indicated that the specific restriction of genomic DNA may happen following the entrance of EcoRI protein most likely into the nucleus of plant cells.

Molecular cloning and expression in Escherichia coli of an active fused Zea mays L. D-amino acid oxidase.

D-Amino acid oxidase (DAAO) is an FAD-dependent enzyme that metabolizes D-amino acids in microbes and animals. However, such ability has not been identified in plants so far. We predicted a complete DAAO coding sequence consisting of 1158 bp and encoding a protein of 386 amino acids.

Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli.

A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length.

Cystatins may confer viral resistance in plants by inhibition of a virus-induced cell death phenomenon in which cysteine proteinases are active: cloning and molecular characterization of a cDNA encoding cysteine-proteinase inhibitor (celostatin) from Celosia cristata (crested cock's comb).

Cystatins (cysteine proteinase inhibitors) have been recently used in plants as antiviral strategy against those viruses whose replication involves cysteine proteinase activity. We proposed an idea that cystatins may confer resistance by inhibition of a virus-induced cell-death phenomenon in which cysteine proteinases are active. To test this idea, a full-length cDNA library was constructed from the preflowering stage of Celosia cristata (crested cock's comb) leaves, and a cDNA clone with cystatin domain was isolated using an oligonucleotide probe designed on the basis of the conserved peptide of plant cystatins.