Recombinant Protein Production and Purification of Insoluble Proteins.

Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The efficient production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the selection of the optimal expression organism and its growth conditions to minimize the formation of insoluble protein aggregates should be done according to the protein characteristics and downstream requirements. Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells.

Advancements in the field of characterization techniques have broadened the opportunities to deepen into nanoparticle production bioprocesses. Gag-based virus-like particles (VLPs) have shown their potential as candidates for recombinant vaccine development. However, comprehensive characterization of the production process is still a requirement to meet the desired critical quality attributes. In this work, the production process of Gag VLPs by baculovirus (BV) infection in the reference High Five and Sf9 insect cell lines is characterized in detail. To this end, the Gag polyprotein was fused in frame to the enhanced green fluorescent protein (eGFP) to favor process evaluation with multiple analytical tools. Tracking of the infection process using confocal microscopy and flow cytometry revealed a pronounced increase in the complexity of High Five over Sf9 cells. Cryogenic transmission electron microscopy (cryo-TEM) characterization determined that changes in cell complexity could be attributed to the presence of occlusion-derived BV in High Five cells, whereas Sf9 cells evidenced a larger proportion of the budded virus phenotype (23-fold). Initial evaluation of the VLP production process using spectrofluorometry showed that higher levels of the Gag-eGFP polyprotein were obtained in High Five cells (3.6-fold). However, comparative analysis based on nanoparticle quantification by flow virometry and nanoparticle tracking analysis (NTA) proved that Sf9 cells were 1.7- and 1.5-fold more productive in terms of assembled VLPs, respectively. Finally, analytical ultracentrifugation coupled to flow virometry evidenced a larger sedimentation coefficient of High Five-derived VLPs, indicating a possible interaction with other cellular compounds. Taken together, these results highlight the combined use of microscopy and flow cytometry techniques to improve vaccine development processes using the insect cell/BV expression vector system. © 2020 International Society for Advancement of Cytometry.

Assembly of histidine-rich protein materials controlled through divalent cations.

Nanostructured protein materials show exciting biomedical applications, since both structure and function can be genetically programmed. In particular, self-assembling histidine-rich proteins benefit from functional plasticity that allows the generation of protein-only nanoparticles for cell targeted drug delivery. However, the rational development of constructs with improved functions is limited by a poor control of the oligomerization process. By exploring cross-interactions between histidine-tagged building blocks, we have identified a critical architectonic role of divalent cations. The obtained data instruct about how histidine-rich protein materials can be assembled, disassembled and reassembled within the nanoscale through the stoichiometric manipulation of divalent ions, in a biochemical approach to biomaterials design. STATEMENT OF SIGNIFICANCE: Divalent metal and non-metal cations such as Ni2+, Cu2+ Ca2+ and Zn2+ have been identified as unexpected molecular tools to control the assembling, disassembling and reassembling of histidine-rich protein materials at the nanoscale. Their stoichiometric manipulation allows generating defined protein-protein cross-molecular contacts between building blocks, for a powerful nano-biochemical manipulation of the material's architecture.

Intrinsic functional and architectonic heterogeneity of tumor-targeted protein nanoparticles.

Self-assembling proteins are gaining attention as building blocks for application-tailored nanoscale materials. This is mostly due to the biocompatibility, biodegradability, and functional versatility of peptide chains. Such a potential for adaptability is particularly high in the case of recombinant proteins, which are produced in living cells and are suitable for genetic engineering. However, how the cell factory itself and the particular protein folding machinery influence the architecture and function of the final material is still poorly explored. In this study we have used diverse analytical approaches, including small-angle X-ray scattering (SAXS) and field emission scanning electron microscopy (FESEM) to determine the fine architecture and geometry of recombinant, tumor-targeted protein nanoparticles of interest as drug carriers, constructed on a GFP-based modular scheme. A set of related oligomers were produced in alternative Escherichia coli strains with variant protein folding networks. This resulted in highly regular populations of morphometric types, ranging from 2.4 to 28 nm and from spherical- to rod-shaped materials. These differential geometric species, whose relative proportions were determined by the features of the producing strain, were found associated with particular fluorescence emission, cell penetrability and receptor specificity profiles. Then, nanoparticles with optimal properties could be analytically identified and further isolated from producing cells for use. The cell's protein folding machinery greatly modulates the final geometry reached by the constructs, which in turn defines the key parameters and biological performance of the material.

Engineering tumor cell targeting in nanoscale amyloidal materials.

Bacterial inclusion bodies are non-toxic, mechanically stable and functional protein amyloids within the nanoscale size range that are able to naturally penetrate into mammalian cells, where they deliver the embedded protein in a functional form. The potential use of inclusion bodies in protein delivery or protein replacement therapies is strongly impaired by the absence of specificity in cell binding and penetration, thus preventing targeting. To address this issue, we have here explored whether the genetic fusion of two tumor-homing peptides, the CXCR4 ligands R9 and T22, to an inclusion body-forming green fluorescent protein (GFP), would keep the interaction potential and the functionality of the fused peptides and then confer CXCR4 specificity in cell binding and further uptake of the materials. The fusion proteins have been well produced in Escherichia coli in their full-length form, keeping the potential for fluorescence emission of the partner GFP. By using specific inhibitors of CXCR4 binding, we have demonstrated that the engineered protein particles are able to penetrate CXCR4+ cells, in a receptor-mediated way, without toxicity or visible cytopathic effects, proving the availability of the peptide ligands on the surface of inclusion bodies. Since no further modification is required upon their purification, the biological production of genetically targeted inclusion bodies opens a plethora of cost-effective possibilities in the tissue-specific intracellular transfer of functional proteins through the use of structurally and functionally tailored soft materials.

CXCR4(+)-targeted protein nanoparticles produced in the food-grade bacterium Lactococcus lactis.

AIM: Lactococcus lactis is a Gram-positive (endotoxin-free) food-grade bacteria exploited as alternative to Escherichia coli for recombinant protein production. We have explored here for the first time the ability of this platform as producer of complex, self-assembling protein materials. MATERIALS & METHODS: Biophysical properties, cell penetrability and in vivo biodistribution upon systemic administration of tumor-targeted protein nanoparticles produced in L. lactis have been compared with the equivalent material produced in E. coli. RESULTS: Protein nanoparticles have been efficiently produced in L. lactis, showing the desired size, internalization properties and biodistribution. CONCLUSION: In vitro and in vivo data confirm the potential and robustness of the production platform, pointing out L. lactis as a fascinating cell factory for the biofabrication of protein materials intended for therapeutic applications.

Tools to cope with difficult-to-express proteins.

The identification of DNA coding sequences contained in the genome of many organisms coupled to the use of high throughput approaches has fueled the field of recombinant protein production. Apart from basic research interests, the growing relevance of this field is highlighted by the global sales of the top ten biopharmaceuticals on the market, which exceeds the trillion USD in a steady increasing tendency. Therefore, the demand of biological compounds seems to have a long run on the market. One of the most popular expression systems is based on Escherichia coli cells which apart from being cost-effective counts with a large selection of resources. However, a significant percentage of the genes of interest are not efficiently expressed in this system, or the expressed proteins are accumulated within aggregates, degraded or lacking the desired biological activity, being finally discarded. In some instances, expressing the gene in a homologous expression system might alleviate those drawbacks but then the process usually increases in complexity and is not as cost-effective as the prokaryotic systems. An increasing toolbox is available to approach the production and purification of those difficult-to-express proteins, including different expression systems, promoters with different strengths, cultivation media and conditions, solubilization tags and chaperone coexpression, among others. However, in most cases, the process follows a non-integrative trial and error strategy with discrete success. This review is focused on the design of the whole process by using an integrative approach, taken into account the accumulated knowledge of the pivotal factors that affect any of the key processes, in an attempt to rationalize the efforts made in this appealing field.

Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles.

A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles.

General introduction: recombinant protein production and purification of insoluble proteins.

Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the selection of the optimal expression organism and the most appropriate growth conditions to minimize the formation of insoluble proteins should be done according to the protein characteristics and downstream requirements. Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.

Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus-like particles produced in Escherichia coli.

Protein nanoparticles such as virus-like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self-assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK(-) E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells.

Sheltering DNA in self-organizing, protein-only nano-shells as artificial viruses for gene delivery.

By recruiting functional domains supporting DNA condensation, cell binding, internalization, endosomal escape and nuclear transport, modular single-chain polypeptides can be tailored to associate with cargo DNA for cell-targeted gene therapy. Recently, an emerging architectonic principle at the nanoscale has permitted tagging protein monomers for self-organization as protein-only nanoparticles. We have studied here the accommodation of plasmid DNA into protein nanoparticles assembled with the synergistic assistance of end terminal poly-arginines (R9) and poly-histidines (H6). Data indicate a virus-like organization of the complexes, in which a DNA core is surrounded by a solvent-exposed protein layer. This finding validates end-terminal cationic peptides as pleiotropic tags in protein building blocks for the mimicry of viral architecture in artificial viruses, representing a promising alternative to the conventional use of viruses and virus-like particles for nanomedicine and gene therapy. FROM THE CLINICAL EDITOR: Finding efficient gene delivery methods still represents a challenge and is one of the bottlenecks to the more widespread application of gene therapy. The findings presented in this paper validate the application of end-terminal cationic peptides as pleiotropic tags in protein building blocks for "viral architecture mimicking" in artificial viruses, representing a promising alternative to the use of viruses and virus-like particles for gene delivery.

Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy.

Traumatic brain injury (TBI) remains as one of the leading causes of mortality and morbidity worldwide and there are no effective treatments currently available. Gene therapy applications have emerged as important alternatives for the treatment of diverse nervous system injuries. New strategies are evolving with the notion that each particular pathological condition may require a specific vector. Moreover, the lack of detailed comparative studies between different vectors under similar conditions hampers the selection of an ideal vector for a given pathological condition. The potential use of lentiviral vectors versus several modular protein-based nanovectors was compared using a controlled cortical impact model of TBI under the same gene therapy conditions. We show that variables such as protein/DNA ratio, incubation volume, and presence of serum or chloroquine in the transfection medium impact on both nanovector formation and transfection efficiency in vitro. While lentiviral vectors showed GFP protein 1 day after TBI and increased expression at 14 days, nanovectors showed stable and lower GFP transgene expression from 1 to 14 days. No toxicity after TBI by any of the vectors was observed as determined by resulting levels of IL-1ß or using neurological sticky tape test. In fact, both vector types induced functional improvement per se.

Improved performance of protein-based recombinant gene therapy vehicles by tuning downstream procedures.

Protein engineering offers a robust platform for the design and production in cell factories of a plethora of protein-based drugs, including nonviral gene therapy vehicles. We have determined here that a protein nanoparticle, formed by highly cationic protein monomers, fails to bind exogenous DNA and to promote detectable gene expression in target cells despite recruiting all the needed functions. Removal of DNA and RNA with nucleases previous to forming complexes with exogenous DNA dramatically enhances the ability of the protein to bind and transfer DNA to target cell nuclei. These data point out contaminant nucleic acids deriving from the cell factory as a major factor impairing the performance of protein-based artificial viruses and stress the need of a nuclease step in the downstream of proteins whose function is based on cationic domains.

Non-amyloidogenic peptide tags for the regulatable self-assembling of protein-only nanoparticles.

Controlling the self-assembling of building blocks as nanoscale entities is a requisite for the generation of bio-inspired vehicles for nanomedicines. A wide spectrum of functional peptides has been incorporated to different types of nanoparticles for the delivery of conventional drugs and nucleic acids, enabling receptor-specific cell binding and internalization, endosomal escape, cytosolic trafficking, nuclear targeting and DNA condensation. However, the development of architectonic tags to induce the self-assembling of functionalized monomers has been essentially neglected. We have examined here the nanoscale architectonic capabilities of arginine-rich cationic peptides, that when displayed on His-tagged proteins, promote their self-assembling as monodisperse, protein-only nanoparticles. The scrutiny of the cross-molecular interactivity cooperatively conferred by poly-arginines and poly-histidines has identified regulatable electrostatic interactions between building blocks that can also be engineered to encapsulate cargo DNA. The combined use of cationic peptides and poly-histidine tags offers an unusually versatile approach for the tailored design and biofabrication of protein-based nano-therapeutics, beyond the more limited spectrum of possibilities so far offered by self-assembling amyloidogenic peptides.

RGD-based cell ligands for cell-targeted drug delivery act as potent trophic factors.

Integrin-binding, Arg-Gly-Asp (RGD)-containing peptides are the most widely used agents to deliver drugs, nanoparticles, and imaging agents. Although in nature, several protein-mediated signal transduction events depend on RGD motifs, the potential of RGD-empowered materials in triggering undesired cell-signaling cascades has been neglected. Using an RGD-functionalized protein nanoparticle, we show here that the RGD motif acts as a powerful trophic factor, supporting extracellular signal-regulated kinase 1/2 (ERK1/2)-linked cell proliferation and partial differentiation of PC12 cells, a neuronlike model. FROM THE CLINICAL EDITOR: This work focuses on RGD peptides, which are among the most commonly used tags for targeted drug delivery. They also promote protoneurite formation and expression of neuronal markers (MAP2) in model PC12 cells, which is an unexpected but relevant event in the functionalization of drugs and their nanocarriers.

Engineered biological entities for drug delivery and gene therapy protein nanoparticles.

The development of genetic engineering techniques has speeded up the growth of the biotechnological industry, resulting in a significant increase in the number of recombinant protein products on the market. The deep knowledge of protein function, structure, biological interactions, and the possibility to design new polypeptides with desired biological activities have been the main factors involved in the increase of intensive research and preclinical and clinical approaches. Consequently, new biological entities with added value for innovative medicines such as increased stability, improved targeting, and reduced toxicity, among others have been obtained. Proteins are complex nanoparticles with sizes ranging from a few nanometers to a few hundred nanometers when complex supramolecular interactions occur, as for example, in viral capsids. However, even though protein production is a delicate process that imposes the use of sophisticated analytical methods and negative secondary effects have been detected in some cases as immune and inflammatory reactions, the great potential of biodegradable and tunable protein nanoparticles indicates that protein-based biotechnological products are expected to increase in the years to come.

Peptide-mediated DNA condensation for non-viral gene therapy.

The construction of non-viral, virus-like vehicles for gene therapy involves the functionalization of multipartite constructs with nucleic acid-binding, cationic agents. Short basic peptides, alone or as fusion proteins, are appropriate DNA binding and condensing elements, whose incorporation into gene delivery vehicles results in the formation of protein-DNA complexes of appropriate size for cell internalization and intracellular trafficking. We review here the most used cationic peptides for artificial virus construction as well as the recently implemented strategies to control the architecture and biological activities of the resulting nanosized particles.