Solid-Solid Interfacial Contact of Tubing Walls Drives Therapeutic Protein Aggregation During Peristaltic Pumping.

Peristaltic pumping during bioprocessing can cause therapeutic protein loss and aggregation during use. Due to the complexity of this apparatus, root-cause mechanisms behind protein loss have been long sought. We have developed new methodologies isolating various peristaltic pump mechanisms to determine their effect on monomer loss. Closed-loops of peristaltic tubing were used to investigate the effects of peristaltic pump parameters on temperature and monomer loss, whilst two mechanism isolation methodologies are used to isolate occlusion and lateral expansion-relaxation of peristaltic tubing. Heat generated during peristaltic pumping can cause heat-induced monomer loss and the extent of heat gain is dependent on pump speed and tubing type. Peristaltic pump speed was inversely related to the rate of monomer loss whereby reducing speed 2.0-fold increased loss rates by 2.0- to 5.0-fold. Occlusion is a parameter that describes the amount of tubing compression during pumping. Varying this to start the contacting of inner tubing walls is a threshold that caused an immediate 20-30% additional monomer loss and turbidity increase. During occlusion, expansion-relaxation of solid-liquid interfaces and solid-solid interface contact of tubing walls can occur simultaneously. Using two mechanisms isolation methods, the latter mechanism was found to be most destructive and a function of solid-solid contact area, where increasing the contact area 2.0-fold increased monomer loss by 1.6-fold. We establish that a form of solid-solid contact mechanism whereby the contact solid interfaces disrupt adsorbed protein films is the root-cause behind monomer loss and protein aggregation during peristaltic pumping.

Biological signal processing filters via engineering allosteric transcription factors.

Signal processing is critical to a myriad of biological phenomena (natural and engineered) that involve gene regulation. Biological signal processing can be achieved by way of allosteric transcription factors. In canonical regulatory systems (e.g., the lactose repressor), an INPUT signal results in the induction of a given transcription factor and objectively switches gene expression from an OFF state to an ON state. In such biological systems, to revert the gene expression back to the OFF state requires the aggressive dilution of the input signal, which can take 1 or more d to achieve in a typical biotic system. In this study, we present a class of engineered allosteric transcription factors capable of processing two-signal INPUTS, such that a sequence of INPUTS can rapidly transition gene expression between alternating OFF and ON states. Here, we present two fundamental biological signal processing filters, BANDPASS and BANDSTOP, that are regulated by D-fucose and isopropyl-ß-D-1-thiogalactopyranoside. BANDPASS signal processing filters facilitate OFF-ON-OFF gene regulation. Whereas, BANDSTOP filters facilitate the antithetical gene regulation, ON-OFF-ON. Engineered signal processing filters can be directed to seven orthogonal promoters via adaptive modular DNA binding design. This collection of signal processing filters can be used in collaboration with our established transcriptional programming structure. Kinetic studies show that our collection of signal processing filters can switch between states of gene expression within a few minutes with minimal metabolic burden-representing a paradigm shift in general gene regulation.

Development of a cell-free protein synthesis system for practical use.

Conventional cell-free protein synthesis systems had been the major platform to study the mechanism behind translating genetic information into proteins, as proven in the central dogma of molecular biology. Albeit being powerful research tools, most of the in vitro methods at the time failed to produce enough protein for practical use. Tremendous efforts were being made to overcome the limitations of in vitro translation systems, though mostly with limited success. While great knowledge was accumulated on the translation mechanism and ribosome structure, researchers rationalized that it may be impossible to fully reconstitute such a complex molecular process in a test tube. This review will examine how we have solved the difficulties holding back progress. Our newly developed cell-free protein synthesis system is based on wheat embryos and has many excellent characteristics, in addition to its high translation activity and robustness. Combined with other novel elementary technologies, we have established cell-free protein synthesis systems for practical use in research and applied sciences.

Engineering with NanoLuc: a playground for the development of bioluminescent protein switches and sensors.

The small engineered luciferase NanoLuc has rapidly become a powerful tool in the fields of biochemistry, chemical biology, and cell biology due to its exceptional brightness and stability. The continuously expanding NanoLuc toolbox has been employed in applications ranging from biosensors to molecular and cellular imaging, and currently includes split complementation variants, engineering techniques for spectral tuning, and bioluminescence resonance energy transfer-based concepts. In this review, we provide an overview of state-of-the-art NanoLuc-based sensors and switches with a focus on the underlying protein engineering approaches. We discuss the advantages and disadvantages of various strategies with respect to sensor sensitivity, modularity, and dynamic range of the sensor and provide a perspective on future strategies and applications.

Programming multi-protein assembly by gene-brush patterns and two-dimensional compartment geometry.

The assembly of protein machines in cells is precise, rapid, and coupled to protein synthesis with regulation in space and time. The assembly of natural and synthetic nanomachines could be similarly controlled by genetic programming outside the cell. Here, we present quasi-two-dimensional (2D) silicon compartments that enable programming of protein assembly lines by local synthesis from surface-immobilized DNA brushes. Using this platform, we studied the autonomous synthesis and assembly of a structural complex from a bacteriophage and a bacterial RNA-synthesizing machine. Local synthesis and surface capture of complexes provided high assembly yield and sensitive detection of spatially resolved assembly intermediates, with the 3D geometry of the compartment and the 2D pattern of brushes dictating the yield and mode of assembly steps. Localized synthesis of proteins in a single gene brush enhances their interactions, and displacement of their genes in separated brushes leads to step-by-step surface assembly. This methodology enables spatial regulation of protein synthesis, and deciphering, reconstruction and design of biological machine assembly lines.

Economics and ecology: Modelling of continuous primary recovery and capture scenarios for recombinant antibody production.

With the maturation of antibody production technologies, both economic optimization and ecological aspects have become important. Continuous downstream processing is a way to reduce the environmental footprint and improve process economics. We compared different primary recovery, capture, and fermentation methods for two output-based antibody production scales: 50 kg/year and 1000 kg/year. In addition, a fixed fermentation volume case of 1000 L was analysed in terms of total cost of goods and process mass intensity as a measure of the environmental footprint. In our scenario, a significant amount of water can be saved in downstream processing when single use equipment is utilized. The overall economic and ecological impact is governed by the product titre in our perfusion (1 g/L) and fed-batch (4 g/L). A low titre in fermentation with similar downstream purification leads to higher process mass intensity and cost of goods due to the higher media demand upstream. The economic perspective for continuous integrated biomanufacturing is very attractive, but environmental consequences should not be neglected. Here, we have shown that perfusion has a higher environmental footprint in the form of water consumption compared to fed-batch. As general guidance to improve process economics, we recommend reducing water consumption.

The Promises and the Challenges of Biotransformations in Microflow.

The challenges of transition toward the postpetroleum world shed light on the biocatalysis as the most sustainable way for the valorization of biobased raw materials. However, its industrial exploitation strongly relies on integration with innovative technologies such as microscale processing. Microflow devices remarkably accelerate biocatalyst screening and engineering, as well as evaluation of process parameters, and intensify biocatalytic processes in multiphase systems. The inherent feature of microfluidic devices to operate in a continuous mode brings additional interest for their use in chemoenzymatic cascade systems and in connection with the downstream processing units. Further steps toward automation and analytics integration, as well as computer-assisted process development, will significantly affect the industrial implementation of biocatalysis and fulfill the promises of the bioeconomy. This review provides an overview of recent examples on implementation of microfluidic devices into various stages of biocatalytic process development comprising ultrahigh-throughput biocatalyst screening, highly efficient biocatalytic process design including specific immobilization techniques for long-term biocatalyst use, integration with other (bio)chemical steps, and/or downstream processing.

High-level expression of Aspergillus niger lipase in Pichia pastoris: Characterization and gastric digestion in vitro.

The low expression level of acidic lipases from Aspergillus sp. remains a major obstacle for their use in industrial applications. In this study, fusion expression with three fusion partners was investigated to enhance the expression level of an acidic lipase from A. niger (ANL) in Pichia pastoris. When fused with a small ubiquitin-related modifier (SUMO), designated SANL, the highest activity reached 960 ±â€¯40 U/mL in a 3 L fermenter, which was 1.85-fold higher than that of the parent ANL. SANL exhibited its maximum activity at pH 2.5 and had lower Km and higher kcat/Km values than those of ANL. In gastrointestinal digestion experiments, SANL was resistant to pepsin and had high hydrolytic activity against triolein from pH 3.0 to 6.0. However, SANL was significantly inhibited by NaTDC above its CMC, which may limit its application for intestinal digestion, but allow it to remain suitable for gastric digestion.

A novel synthetic trivalent single chain variable fragment (tri-scFv) construction platform based on the SpyTag/SpyCatcher protein ligase system.

BACKGROUND: Advances in antibody engineering provide strategies to construct recombinant antibody-like molecules with modified pharmacokinetic properties. Multermerization is one strategy that has been used to produce antibody-like molecules with two or more antigen binding sites. Multimerization enhances the functional affinity (avidity) and can be used to optimize size and pharmacokinetic properties. Most multimerization strategies involve genetically fusing or non-covalently linking antibody fragments using oligomerization domains. Recent studies have defined guidelines for producing antibody-like molecules with optimal tumor targeting properties, which require intermediates size (70-120 kDa) and bi- or tri-valency. RESULTS: We described a highly modular antibody-engineering platform for rapidly constructing synthetic, trivalent single chain variable fragments (Tri-scFv) using the SpyCatcher/SpyTag protein ligase system. We used this platform to construct an anti-human epidermal growth factor receptor 3 (HER3) Tri-scFv. We generated the anti-HER3 Tri-scFv by genetically fusing a SpyCatcher to the C-terminus of an anti-HER3 scFv and ligating it to a synthetic Tri-SpyTag peptide. The anti-HER3 Tri-scFv bound recombinant HER3 with an apparent KD of 2.67 nM, which is approximately 12 times lower than the KD of monomeric anti-HER3 scFv (31.2 nM). Anti-HER3 Tri-scFv also bound endogenous cell surface expressed HER3 stronger than the monomer anti-HER3 scFv. CONCLUSION: We used the SpyTag/SpyCatcher protein ligase system to ligate anti-HER3 scFv fused to a SpyCatcher at its C-termini to a Tri-SpyTag to construct Tr-scFv. This system allowed the construction of a Tri-scFv with all the scFv antigen-binding sites pointed outwards. The anti-HER3 Tri-scFv bound recombinant and endogenously expressed HER3 with higher functional affinity (avidity) than the monomeric anti-HER3 scFv. The Tri-scFv had the size, valency, and functional affinity that are desired for therapeutic and imaging applications. Use of the SpyTag/SpyCatcher protein ligase system allows Tri-scFvs to be rapidly constructed in a simple, modular manner, which can be easily applied to scFvs or other antibody fragments targeting other antigens.

Experimental Protocols for Generating Focused Mutant Libraries and Screening for Thermostable Proteins.

Many proteins are rapidly deactivated when exposed to high or even ambient temperatures. This cannot only impede the study of a particular protein, but also is one of the major reasons why enzyme catalysis is still widely unable to compete with established chemical processes. Furthermore, differences in protein stability are a challenge in synthetic biology, when individual modules prove to be incompatible. The targeted stabilization of proteins can overcome these hurdles, and protein engineering techniques are more and more reliably supported by computational chemistry tools. Accordingly, algorithms to predict the differences in folding energy of a mutant compared to the wild-type, ΔΔGfold, are used in the highly successful FRESCO workflow. The resulting single mutant prediction library consists typically of a few hundred amino acid exchanges, and after combining the most successful hits we so far obtained stabilized mutants which exhibited increases in apparent melting temperature of 20-35°C and showed vastly increased half-lives, as well as resistance to cosolvents. Here, we report a detailed protocol to generate these mutant libraries experimentally, covering the entire workflow from primer design, through mutagenesis, protein production and screening, to mutation combination strategies. The individual parts of the method are furthermore applicable to many other scenarios besides protein stabilization, and these protocols are valuable for any project requiring individual or semi high-throughput site-directed mutagenesis, protein expression and purification, or generation of mutant combination libraries.

Cobalamin-Dependent C-Methyltransferases From Marine Microbes: Accessibility via Rhizobia Expression.

Cobalamin-dependent radical S-adenosylmethionine (rSAM) methyltransferases catalyze chemically challenging methylation reactions on diverse natural products at unactivated carbon centers. In vivo reconstitution and biosynthetic studies of natural product gene clusters encoding these enzymes are often severely limited by ineffective heterologous expression hosts, including the otherwise versatile Escherichia coli. In this chapter, we describe the use of rhizobia bacteria as effective expression hosts for cobalamin-dependent rSAM C-methyltransferases. We chose the natural product pathway encoding the heavily modified cytotoxic peptides, the polytheonamides, as our model pathway due to the presence of two methyltransferases responsible for a total of 17 C-methylations. Detailed protocols are given for vector construction, transformation, and heterologous expression in Rhizobium leguminosarum bv. viciae 3841. Additional methods pertaining to analytical separation and mass spectrometric analysis of modified peptides are also entailed. As genomics continues to uncover new enzymes and pathways from unknown and uncultivated microbes, use of metabolically distinct heterologous expression hosts like rhizobia will be a necessary tool to unravel the catalytic and metabolic diversity of marine microbial life.

Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential.

Processes involved in the functional formation of prokaryotic membrane proteins have remained elusive. Here, we developed a new in vitro membrane protein expression system to detect nascent activities of the KcsA potassium channel in lipid bilayers under an applied membrane potential. The channel was synthesized using a reconstituted Escherichia coli-based in vitro transcription/translation system (IVTT) in a water-in-oil droplet lined by a membrane. The synthesized channels spontaneously incorporated into the membrane even without the translocon machinery (unassisted pathway) and formed functional channels with the correct orientation. The single-channel current of the first appearing nascent channel was captured, followed by the subsequent appearance of multiple channels. Notably, the first appearance time shortened substantially as the membrane potential was hyperpolarized. Under a steadily applied membrane potential, this system serves as a production line of membrane proteins via the unassisted pathway, mimicking the bacterial synthetic membrane.

Intracellular drug delivery: Potential usefulness of engineered Shiga toxin subunit B for targeted cancer therapy.

A treasure trove of intracellular cancer drug targets remains hidden behind cell membranes. However, engineered pathogen-derived toxins such as Shiga toxins can deliver small or macromolecular drugs to specific intracellular organelles. After binding to ganglioglobotriaosylceramide (Gb3, CD77), the non-toxic subunit B (StxB) of the Shiga-holotoxin is endocytosed and delivers its payload by a unique retrograde trafficking pathway via the endoplasmic reticulum to the cytosol. This review provides an overview of biomedical applications of StxB-based drug delivery systems in targeted cancer diagnosis and therapy. Biotechnological production of the Stx-material is discussed from the perspective of developing efficacious and safe therapeutics.

Leveraging single-pass tangential flow filtration to enable decoupling of upstream and downstream monoclonal antibody processing.

Decoupling upstream and downstream operations in biopharmaceutical production could enable more flexible manufacturing operations and could allow companies to leverage strategic or financial benefits that would be otherwise unattainable. A decoupling process was developed and scaled up utilizing single-pass tangential flow filtration for volume reduction, followed by bulk freezing in single-use bags prior to purification. Single-pass tangential flow filtration can be used to continuously concentrate harvested cell culture fluid, reducing the volume by 15-25× with a step yield of >96%. These concentration factors were reproduced with a second product, indicating that the process could be amenable to platform processes. Experimental data indicate that the product tested was stable for at least one year at -40 or -70°C. The concentration of the harvested cell culture fluid-either with or without a subsequent period of frozen storage-had no impact on the product quality attributes that were tested. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:405-411, 2018.

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Optimizing cell-free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations.

Cell-free protein synthesis (CFPS) is an ideal platform for rapid and convenient protein production. However, bioreactor design remains a critical consideration in optimizing protein expression. Using turbo green fluorescent protein (tGFP) as a model, we tracked small molecule components in a Chinese Hamster Ovary (CHO) CFPS system to optimize protein production. Here, three bioreactors in continuous-exchange cell-free (CECF) format were characterized. A GFP optical sensor was built to monitor the product in real-time. Mass transfer of important substrate and by-product components such as nucleoside triphosphates (NTPs), creatine, and inorganic phosphate (Pi) across a 10-kDa MWCO cellulose membrane was calculated. The highest efficiency measured by tGFP yields were found in a microdialysis device configuration; while a negative effect on yield was observed due to limited mass transfer of NTPs in a dialysis cup configuration. In 24-well plate high-throughput CECF format, addition of up to 40 mM creatine phosphate in the system increased yields by up to ∼60% relative to controls. Direct ATP addition, as opposed to creatine phosphate addition, negatively affected the expression. Pi addition of up to 30 mM to the expression significantly reduced yields by over ∼40% relative to controls. Overall, data presented in this report serves as a valuable reference to optimize the CHO CFPS system for next-generation bioprocessing. Biotechnol. Bioeng. 2017;114: 1478-1486. © 2017 Wiley Periodicals, Inc.

Engineering Halomonas spp. as A Low-Cost Production Host for Production of Bio-surfactant Protein PhaP.

Halomonas spp. have been studied as a low cost production host for producing bulk materials such as polyhydroxyalkanoates (PHA) bioplastics, since they are able to grow at high pH and high NaCl concentration under unsterile and continuous conditions without microbial contamination. In this paper, Halomonas strain TD is used as a host to produce a protein named PHA phasin or PhaP which has a potential to be developed into a bio-surfactant. Four Halomonas TD expression strains are constructed based on a strong T7-family expression system. Of these, the strain with phaC deletion and chromosomal expression system resulted in the highest production of PhaP in soluble form, reaching 19% of total cellular soluble proteins and with a yield of 1.86 g/L in an open fed-batch fermentation process. A simple "heat lysis and salt precipitation" method is applied to allow rapid PhaP purification from a mixture of cellular proteins with a PhaP recovery rate of 63%. It clearly demonstrated that Halomonas TD could be used for high yield expression of a bio-surfactant protein PhaP for industrial application in an economical way.

Secreted expression of truncated capsid protein from porcine circovirus type 2 in Pichia pastoris.

OBJECTIVE: To achieve secreted expression of the truncated capsid protein from porcine circovirus type 2 (PCV2) in Pichia pastoris. RESULTS: A truncated cap gene (tcap) with a deleted N-terminal nuclear localization signal was optimized and synthesized. Effective secreted expression was achieved in P. pastoris GS115. The high-productive recombinant strain for tCap was grown in a 5 l bioreactor and the productivity of tCap in supernatant reached 250 µg/ml. Furthermore, serum antibody test demonstrated that adjuvant-assisting tCap induced a significant increase of specific PCV2-Cap antibody over time in mice and a similar antibody level in pigs compared with a commercial Cap-based subunit vaccine. CONCLUSION: This work establishes a secreted expression strategy in P. pastoris for the production of PCV2 Cap with superior bioactivity, and this strategy might provide potential uses in developing Cap-based subunit vaccine in the future.

Fully automatized high-throughput enzyme library screening using a robotic platform.

A fully automatized robotic platform has been established to facilitate high-throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiter plate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and multiple assay screenings. The performance of this system has been exemplified for four enzyme classes important for biocatalysis such as Baeyer-Villiger monooxygenase, transaminase, dehalogenase and acylase in the high-throughput screening of various mutant libraries. This allowed the identification of novel enzyme variants in a sophisticated and highly reliable manner. Furthermore, the detailed optimization protocols should enable other researchers to adapt and improve their methods. Biotechnol. Bioeng. 2016;113: 1421-1432. © 2016 Wiley Periodicals, Inc.