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We produced human growth hormone (hGH), a protein that stimulates growth and cell reproduction, in genetically engineered soybean [Glycine max (L.) Merrill] seeds. Utilising the alpha prime (α') subunit of β-conglycinin tissue-specific promoter from soybean and the α-Coixin signal peptide from Coix lacryma-jobi, we obtained transgenic soybean lines that expressed the mature form of hGH in their seeds. Expression levels of bioactive hGH up to 2.9% of the total soluble seed protein content (corresponding to approximately 9 g kg(-1)) were measured in mature dry soybean seeds. The results of ultrastructural immunocytochemistry assays indicated that the recombinant hGH in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Specific bioassays demonstrated that the hGH expressed in the soybean seeds was fully active. The recombinant hGH protein sequence was confirmed by mass spectrometry characterisation. These results demonstrate that the utilisation of tissue-specific regulatory sequences is an attractive and viable option for achieving high-yield production of recombinant proteins in stable transgenic soybean seeds.

The use of mass spectrometry to identify recombinant proteins that are expressed in total soluble proteins (TSPs) from plant extracts is necessary to accelerate further processing steps. For example, the method consists of TSP sample preparation and trypsin digestion prior to the preliminary characterization using nanoUPLC-MS(E) analysis of the recombinant proteins that are expressed in TSP samples of transgenic soybean seeds. A TSP sample as small as 50 μg can be effectively analyzed. In this study, transgenic soybean seeds that expressed recombinant cancer testis antigen (CTAG) were used. The procedure covered 30% of the protein sequence and was quantified at 0.26 ng, which corresponded to 0.1% of the TSP sample. A comparative proteomic profile was generated by the comparison of a negative control and sample that showed a unique expression pattern of CTAG in a transgenic line. The experimental data from the TSP extraction, sample preparation and data analysis are discussed herein.

Antimicrobial peptides are an abundant group of proteinaceous compounds widely produced in the plant kingdom. Among them, the γ‐thionin family, also known as plant defensins, represents one typical family and comprises low molecular mass cysteine‐rich proteins, usually cationic and distributed in different plant tissues. Here, we report the purification and characterization of a novel γ‐thionin from cowpea seeds (Vigna unguiculata), named Cp‐thionin II, with bactericidal activity against Gram‐positive and Gram‐negative bacteria. Once the primary structure was elucidated, molecular modelling experiments were used to investigate the multimerization and mechanism of action of plant γ‐thionins. Furthermore, Cp‐thionin II was also localized in different tissues in cowpea seedlings during germination in contrasting conditions, to better understand the plant protection processes. The use of plant defensins in the construction of transgenic plants and also in the production of novel drugs with activity against human pathogens is discussed.

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Plant defensins are small cysteine-rich proteins commonly synthesized in plants, encoded by large multigene families. Most plant defensins that have been characterized to date show potent antifungal and/or bactericidal activities. This report describes VuD1, an unusual defensin that is able to inhibit insect-pest alpha-amylases. VuD1 was cloned from cowpea (Vigna unguiculata) seeds and expressed in a heterologous system. Inhibitory enzyme assays showed that VuD1 efficiently inhibits alpha-amylases from the weevils Acanthoscelides obtectus and Zabrotes subfasciatus, caused low inhibition toward mammalian enzymes and was unable to inhibit the alpha-amylases from Callosobruchus maculatus and Aspergillus fumigatus. To shed some light over the mechanism of action of VuD1, molecular modeling analyses were performed, revealing that the N-terminus of the molecule is responsible for binding with the active site of weevil enzymes. Moreover, models of VuD1 and mammalian enzymes were also generated to elucidate the specificity mechanisms. The data presented herein suggests that this defensin has potential application in the development of transgenic plants for insect pest control.

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

SummaryThere is an urgent need to provide effective anti‐HIV microbicides to resource‐poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant cyanovirin‐N (rCV‐N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV‐N was isolated with a yield of 350 μg/g of dry seed weight. The observed amino acid sequence of rCV‐N matched the expected sequence of native CV‐N, as did the mass of rCV‐N (11 009 Da). Purified rCV‐N from soya is active in anti‐HIV assays with an EC50 of 0.82–2.7 nM (compared to 0.45–1.8 nM for E. coli‐produced CV‐N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV‐N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti‐HIV microbicide development.