Plastid transformation: advances and challenges for its implementation in agricultural crops

Chloroplast biotechnology has emerged as a promissory platform for the development of modified plants to express products aimed mainly at the pharmaceutical, agricultural, and energy industries. This technology's high value is due to its high capacity for the mass production of proteins. Moreover, the interest in chloroplasts has increased because of the possibility of expressing multiple genes in a single transformation event without the risk of epigenetic effects. Although this technology solves several problems caused by nuclear genetic engineering, such as turning plants into safe bio-factories, some issues must still be addressed in relation to the optimization of regulatory regions for efficient gene expression, cereal transformation, gene expression in non-green tissues, and low transformation efficiency. In this article, we provide information on the transformation of plastids and discuss the most recent achievements in chloroplast bioengineering and its impact on the biopharmaceutical and agricultural industries; we also discuss new tools that can be used to solve current challenges for their successful establishment in recalcitrant crops such as monocots.

Structure-Function Relationship Study of a Secretory Amoebic Phosphatase: A Computational-Experimental Approach.

Phosphatases are hydrolytic enzymes that cleave the phosphoester bond of numerous substrates containing phosphorylated residues. The typical classification divides them into acid or alkaline depending on the pH at which they have optimal activity. The histidine phosphatase (HP) superfamily is a large group of functionally diverse enzymes characterized by having an active-site His residue that becomes phosphorylated during catalysis. HP enzymes are relevant biomolecules due to their current and potential application in medicine and biotechnology. Entamoeba histolytica, the causative agent of human amoebiasis, contains a gene (EHI_146950) that encodes a putative secretory acid phosphatase (EhHAPp49), exhibiting sequence similarity to histidine acid phosphatase (HAP)/phytase enzymes, i.e., branch-2 of HP superfamily. To assess whether it has the potential as a biocatalyst in removing phosphate groups from natural substrates, we studied the EhHAPp49 structural and functional features using a computational-experimental approach. Although the combined outcome of computational analyses confirmed its structural similarity with HP branch-2 proteins, the experimental results showed that the recombinant enzyme (rEhHAPp49) has negligible HAP/phytase activity. Nonetheless, results from supplementary activity evaluations revealed that rEhHAPp49 exhibits Mg2+-dependent alkaline pyrophosphatase activity. To our knowledge, this study represents the first computational-experimental characterization of EhHAPp49, which offers further insights into the structure-function relationship and the basis for future research.

Novel Genetic Constructs for Production of Recombinant HTLV-1/2 Antigens and Evaluation of Their Reactivity to Plasma Samples from HTLV-1-Infected Patients.

Human T-cell leukemia virus type 1 (HTLV-1) can cause life-threatening diseases for which there are no effective treatments. Prevention of HTLV-1 infection requires massive testing of pregnant women, blood for transfusion, and organs for transplantation, as well as safe sex. In this context, serological assays are widely used for monitoring HTLV-1 infections. Despite the necessity for recombinant antigens to compose serological tests, there is little information available on procedures to produce recombinant HTLV-1/2 antigens for serological diagnostic purposes. In this work, we tested a series of genetic constructions to select those more amenable for production in bacterial systems. To overcome the constraints in expressing sections of viral envelope proteins in bacteria, we have used the p24 segment of the gag protein as a scaffold to display the immunogenic regions of gp46 and gp21. Nine recombinant antigenic proteins derived from HTLV-1 and five derived from HTLV-2 were successfully purified. The HTLV-1 antigens showed high efficiency in discriminating HTLV-positive samples from HTLV-negative samples using enzyme-linked immunosorbent assays. Interestingly, HTLV-1-positive samples showed a high level of cross-reaction with HTLV-2 antigens. This finding is explained by the high sequence conservation between the structural proteins of these two highly related viruses. In summary, the results presented in this work provide a detailed description of the methods used to produce recombinant HTLV-1 and HTLV-2 antigens, and they demonstrate that the HTLV-1 antigens show strong potential for serological diagnosis of HTLV-1 infections.

Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells.

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.

New tools for recombinant protein production in Escherichia coli: A 5-year update.

The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost-effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.

Systematic Comparison of Strategies to Achieve Soluble Expression of Plasmodium falciparum Recombinant Proteins in E. coli.

Constructs containing partial coding sequences of myosin A, myosin B, and glideosome-associated protein (50 kDa) of Plasmodium falciparum were used to challenge several strategies designed in order to improve the production and solubility of recombinant proteins in Escherichia coli. Assays were carried out inducing expression in a late log phase culture, optimizing the inductor concentration, reducing the growth temperature for induced cultures, and supplementing additives in the lysis buffer. In addition, recombinant proteins were expressed as fusion proteins with three different tags (6His, GST, and MBP) in four different E. coli strains. We found that the only condition that consistently produced soluble proteins was the use of MBP as a fusion tag, which became a valuable tool for detecting the proteins used in this study and did not caused any interference in protein-protein interaction assays (Far Western Blot). Besides, we found that BL21-pG-KJE8 strain did not improve the solubility of any of the recombinant protein produced, while the BL21-CodonPlus(DE3)-RIL strain improved the expression of some of them independent of the rare codon content. Proteins with rare codons occurring at high frequencies (¼ 10%) were expressed efficiently in strains that do not supplement tRNAs for these triplets.

DROSOPHILA S2 cell culture in a WAVE Bioreactor: potential for scaling up the production of the recombinant rabies virus glycoprotein.

The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 × 105 cells/mL in culture medium (Sf900-III) induced with solution of CuSO4 (0.7 mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72 h) and temperature (28 °C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor™ 2/10 using a 2 L Cellbag. The results in Schott flasks and in WAVE Bioreactor™ were very similar, yielding a maximum titer of rRVGP above of 1 mg.L-1. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.

Drosophila S2 cell culture in a wave bioreactor: potential for scaling up the production of the recombinant rabies virus glycoprotein

The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 x 10(5) cells/mL in culture medium (Sf900-III) induced with solution of CuSO4 (0.7 mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72 h) and temperature (28 degrees C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor (TM) 2/10 using a 2 L Cellbag. The results in Schott flasks and inWAVE Bioreactor (TM) were very similar, yielding a maximum titer of rRVGP above of 1 mg.L-1. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.

Production of recombinant coagulation factors: Are humans the best host cells?

The main treatment option for Hemophilia A/B patients involves the administration of recombinant coagulation factors on-demand or in a prophylactic approach. Despite the safety and efficacy of this replacement therapy, the development of antibodies against the coagulation factor infused, which neutralize the procoagulant activity, is a severe complication. The production of recombinant coagulation factors in human cell lines is an efficient approach to avoid such complication. Human cell lines can produce recombinant proteins with post translation modifications more similar to their natural counterpart, reducing potential immunogenic reactions. This review provides a brief overview of the most important characteristics of recombinant FVIII and FIX products available on the market and the improvements that have recently been achieved by the production using human cell lines.

A constitutive expression system for Pichia pastoris based on the PGK1 promoter.

OBJECTIVES: To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter. RESULTS: P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants. CONCLUSIONS: A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

Improvement of recombinant protein production by an anti-apoptotic protein from hemolymphof Lonomia obliqua

Apoptosis is a major problem in animalcell culture during production of biopharmaceuticals, such as recombinant proteins or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotaviruslike-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection also induces host celldeath. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein productionof BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph of Lonomia obliqua were able toprotect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability and productivity of recombinantprotein in BEVS/IC system.