Synthetic Tuning of Domain Stoichiometry in Nanobody-Enzyme Megamolecules.

This paper presents a method to synthetically tune atomically precise megamolecule nanobody-enzyme conjugates for prodrug cancer therapy. Previous efforts to create heterobifunctional protein conjugates suffered from heterogeneity in domain stoichiometry, which in part led to the failure of antibody-enzyme conjugates in clinical trials. We used the megamolecule approach to synthesize anti-HER2 nanobody-cytosine deaminase conjugates with tunable numbers of nanobody and enzyme domains in a single, covalent molecule. Linking two nanobody domains to one enzyme domain improved avidity to a human cancer cell line by 4-fold but did not increase cytotoxicity significantly due to lowered enzyme activity. In contrast, a megamolecule composed of one nanobody and two enzyme domains resulted in an 8-fold improvement in the catalytic efficiency and increased the cytotoxic effect by over 5-fold in spheroid culture, indicating that the multimeric structure allowed for an increase in local drug activation. Our work demonstrates that the megamolecule strategy can be used to study structure-function relationships of protein conjugate therapeutics with synthetic control of protein domain stoichiometry.

Long-Range Energy Transfer in Protein Megamolecules.

In this investigation, we report evidence for energy transfer in new protein-based megamolecules with tunable distances between donor and acceptor fluorescent proteins. The megamolecules used in this work are monodisperse oligomers, with molecular weights of ∼100-300 kDa and lengths of ∼5-20 nm, and are precisely defined structures of fusion protein building blocks and covalent cross-linkers. Such structures are promising because the study of energy transfer in protein complexes is usually difficult in this long length regime due to synthetic limitations. We incorporated fluorescent proteins into the megamolecule structure and varied the separation distance between donor and acceptor by changing the length of the cross-linker in dimer conjugates and inserting nonfluorescent spacer proteins to create oligomers. Two-photon absorption measurements demonstrated strong coupling between donor and acceptor dipoles in the megamolecules. For the dimer systems, no effect of the cross-linker length on energy transfer efficiency was observed with the steady-state fluorescence investigation. However, for the same dimer conjugates, energy transfer rates decreased upon increasing cross-linker length, as evaluated by fluorescence up-conversion. Molecular dynamics simulations were used to rationalize the results, providing quantitative agreement between measured and calculated energy transfer lengths for steady-state results, and showing that the differences between the time-resolved and steady-state measurements arise from the long time scale for large-scale fluctuations in the megamolecule structure. Our results show an increase in energy transfer length with increasing megamolecule size. This is evidence for long-range energy transfer in large protein megamolecules.

An estimate is worth about a thousand experiments: using order-of-magnitude estimates to identify cellular engineering targets.

Biotechnological processes use microbes to convert abundant molecules, such as glucose, into high-value products, such as pharmaceuticals, commodity and fine chemicals, and energy. However, from the outset of the development of a new bioprocess, it is difficult to determine the feasibility, expected yields, and targets for engineering. In this review, we describe a methodology that uses rough estimates to assess the feasibility of a process, approximate the expected product titer of a biological system, and identify variables to manipulate in order to achieve the desired performance. This methodology uses estimates from literature and biological intuition, and can be applied in the early stages of a project to help plan future engineering. We highlight recent literature examples, as well as two case studies from our own work, to demonstrate the use and power of rough estimates. Describing and predicting biological function using estimates guides the research and development phase of new bioprocesses and is a useful first step to understand and build a new microbial factory.

Type III secretion as a generalizable strategy for the production of full-length biopolymer-forming proteins.

Biopolymer-forming proteins are integral in the development of customizable biomaterials, but recombinant expression of these proteins is challenging. In particular, biopolymer-forming proteins have repetitive, glycine-rich domains and, like many heterologously expressed proteins, are prone to incomplete translation, aggregation, and proteolytic degradation in the production host. This necessitates tailored purification processes to isolate each full-length protein of interest from the truncated forms as well as other contaminating proteins; owing to the repetitive nature of these proteins, the truncated polypeptides can have very similar chemistry to the full-length form and are difficult to separate from the full-length protein. We hypothesized that bacterial expression and secretion would be a promising alternative option for biomaterials-forming proteins, simplifying isolation of the full-length target protein. By using a selective secretion system, truncated forms of the protein are not secreted and thus are not found in the culture harvest. We show that a synthetically upregulated type III secretion system leads to a general increase in secretion titer for each protein that we tested. Moreover, we observe a substantial enhancement in the homogeneity of full-length forms of pro-resilin, tropo-elastin crosslinking domains, and silk proteins produced in this manner, as compared with proteins purified from the cytosol. Secretion via the type III apparatus limits co-purification of truncated forms of the target protein and increases protein purity without extensive purification steps. Demonstrating the utility of such a system, we introduce several modifications to resilin-based peptides and use an un-optimized, single-column process to purify these proteins. The resulting materials are of sufficiently high quantity and yield for the production of antimicrobial hydrogels with highly reproducible rheological properties. The ease of this process and its applicability to an array of engineered biomaterial-forming peptides lend support for the application of bacterial expression and secretion for other proteins that are traditionally difficult to express and isolate from the bacterial cytoplasm. Biotechnol. Bioeng. 2016;113: 2313-2320. © 2015 Wiley Periodicals, Inc.

Proteins adopt functionally active conformations after type III secretion.

BACKGROUND: Bacterial production of natively folded heterologous proteins by secretion to the extracellular space can improve protein production by simplifying purification and enabling continuous processing. In a typical bacterial protein production process, the protein of interest accumulates in the cytoplasm of the cell, requiring cellular lysis and extensive purification to separate the desired protein from other cellular constituents. The type III secretion system of Gram-negative bacteria is used to secrete proteins from the cytosol to the extracellular space in one step, but proteins must unfold during translocation, necessitating the folding of secreted proteins in the extracellular space for an efficient production process. We evaluated type III secretion as a protein production strategy by characterizing and quantifying the extent of correct folding after secretion. RESULTS: We probed correct folding by assaying the function after secretion of two enzymes-beta-lactamase and alkaline phosphatase-and one single-chain variable fragment of an antibody. Secreted proteins are correctly folded and functional after unfolding, secretion, and refolding in the extracellular space. Furthermore, structural and chemical features required for protein function, such as multimerization and disulfide bond formation, are evident in the secreted protein samples. Finally, the concentration of NaCl in the culture media affects the folding efficiency of secreted proteins in a protein-specific manner. CONCLUSIONS: In the extracellular space, secreted proteins are able to fold to active conformations, which entails post-translational modifications including: folding, multimerization, acquisition of metal ion cofactors, and formation of disulfide bonds. Further, different proteins have different propensities to refold in the extracellular space and are sensitive to the chemical environment in the extracellular space. Our results reveal strategies to control the secretion and correct folding of diverse target proteins during bacterial cell culture.

Using transcriptional control to increase titers of secreted heterologous proteins by the type III secretion system.

The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28±9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant.

Directed evolution of brain-derived neurotrophic factor for improved folding and expression in Saccharomyces cerevisiae.

Brain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employing Saccharomyces cerevisiae display and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF.

Design of a mucin-selective protease for targeted degradation of cancer-associated mucins.

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

A machine learning strategy for the identification of key in silico descriptors and prediction models for IgG monoclonal antibody developability properties.

Identification of favorable biophysical properties for protein therapeutics as part of developability assessment is a crucial part of the preclinical development process. Successful prediction of such properties and bioassay results from calculated in silico features has potential to reduce the time and cost of delivering clinical-grade material to patients, but nevertheless has remained an ongoing challenge to the field. Here, we demonstrate an automated and flexible machine learning workflow designed to compare and identify the most powerful features from computationally derived physiochemical feature sets, generated from popular commercial software packages. We implement this workflow with medium-sized datasets of human and humanized IgG molecules to generate predictive regression models for two key developability endpoints, hydrophobicity and poly-specificity. The most important features discovered through the automated workflow corroborate several previous literature reports, and newly discovered features suggest directions for further research and potential model improvement.

Immunosuppressive glycoproteins associate with breast tumor fibrosis and aggression.

Tumors feature elevated sialoglycoprotein content. Sialoglycoproteins promote tumor progression and are linked to immune suppression via the sialic acid-Siglec axis. Understanding factors that increase sialoglycoprotein biosynthesis in tumors could identify approaches to improve patient response to immunotherapy. We quantified higher levels of sialoglycoproteins in the fibrotic regions within human breast tumor tissues. Human breast tumor subtypes, which are more fibrotic, similarly featured increased sialoglycoprotein content. Further analysis revealed the breast cancer cells as the primary cell type synthesizing and secreting the tumor tissue sialoglycoproteins and confirmed that the more aggressive, fibrotic breast cancer subtypes expressed the highest levels of sialoglycoprotein biosynthetic genes. The more aggressive breast cancer subtypes also featured greater infiltration of immunosuppressive SIGLEC7, SIGLEC9, and SIGLEC10-pos myeloid cells, indicating that triple-negative breast tumors had higher expression of both immunosuppressive Siglec receptors and their cognate ligands. The findings link sialoglycoprotein biosynthesis and secretion to tumor fibrosis and aggression in human breast tumors. The data suggest targeting of the sialic acid-Siglec axis may comprise an attractive therapeutic target particularly for the more aggressive HER2+ and triple-negative breast cancer subtypes.

Leveraging microenvironmental synthetic lethalities to treat cancer.

Treatment resistance leads to cancer patient mortality. Therapeutic approaches that employ synthetic lethality to target mutational vulnerabilities in key tumor cell signaling pathways have proven effective in overcoming therapeutic resistance in some cancers. Yet, tumors are organs composed of malignant cells residing within a cellular and noncellular stroma. Tumor evolution and resistance to anticancer treatment are mediated through a dynamic and reciprocal dialogue with the tumor microenvironment (TME). Accordingly, expanding tumor cell synthetic lethality to encompass contextual synthetic lethality has the potential to eradicate tumors by targeting critical TME circuits that promote tumor progression and therapeutic resistance. In this Review, we summarize current knowledge about the TME and discuss its role in treatment. We outline the concept of tumor cell-specific synthetic lethality and describe therapeutic approaches to expand this paradigm to leverage TME synthetic lethality to improve cancer therapy.

Concepts of extracellular matrix remodelling in tumour progression and metastasis.

Tissues are dynamically shaped by bidirectional communication between resident cells and the extracellular matrix (ECM) through cell-matrix interactions and ECM remodelling. Tumours leverage ECM remodelling to create a microenvironment that promotes tumourigenesis and metastasis. In this review, we focus on how tumour and tumour-associated stromal cells deposit, biochemically and biophysically modify, and degrade tumour-associated ECM. These tumour-driven changes support tumour growth, increase migration of tumour cells, and remodel the ECM in distant organs to allow for metastatic progression. A better understanding of the underlying mechanisms of tumourigenic ECM remodelling is crucial for developing therapeutic treatments for patients.

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding.

Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT's activity than previously proposed.

Photoactivatable Reaction for Covalent Nanoscale Patterning of Multiple Proteins.

This article describes a photochemical approach for independently patterning multiple proteins to an inert substrate, particularly for studies of cell adhesion. A photoactivatable chloropyrimidine ligand was employed for covalent immobilization of SnapTag fusion proteins on self-assembled monolayers of alkanethiolates on gold. A two-step procedure was used: first, patterned UV illumination of the surface activated protein capture ligands, and second, incubation with a SnapTag fusion protein bound to the surface in illuminated regions. Two different fluorescent proteins were patterned in registry with features of 400 nm in size over a 1 mm2 area. An example is given wherein an anti-carcinoembryonic antigen (anti-CEA) scFv antibody was patterned to direct the selective attachment of a human cancer cell line that express the CEA antigen. This method enables the preparation of surfaces with control over the density and activity of independently patterned proteins.

Use of Transcriptional Control to Increase Secretion of Heterologous Proteins in T3S Systems.

Heterologous proteins can be produced in a bacterial host and purified from the cellular constituents. Secretion of the protein of interest to the extracellular space simplifies the purification process and is thought to alleviate toxicity problems associated with intracellular accumulation of the protein of interest. In this protocol, we describe a strategy to engineer protein secretion in a bacterial culture using transcriptional control. The transcription factor HilA is inducibly produced to control production of the secretion machine, and in turn signals the production and secretion of a protein of interest. This allows for high titer of secreted protein in optimized culturing conditions and the effect is observed with all proteins tested.

Pro-region engineering for improved yeast display and secretion of brain derived neurotrophic factor.

Brain derived neurotrophic factor (BDNF) is a promising therapeutic candidate for a variety of neurological diseases. However, it is difficult to produce as a recombinant protein. In its native mammalian context, BDNF is first produced as a pro-protein with subsequent proteolytic removal of the pro-region to yield mature BDNF protein. Therefore, in an attempt to improve yeast as a host for heterologous BDNF production, the BDNF pro-region was first evaluated for its effects on BDNF surface display and secretion. Addition of the wild-type pro-region to yeast BDNF production constructs improved BDNF folding both as a surface-displayed and secreted protein in terms of binding its natural receptors TrkB and p75, but titers remained low. Looking to further enhance the chaperone-like functions provided by the pro-region, two rounds of directed evolution were performed, yielding mutated pro-regions that further improved the display and secretion properties of BDNF. Subsequent optimization of the protease recognition site was used to control whether the produced protein was in pro- or mature BDNF forms. Taken together, we have demonstrated an effective strategy for improving BDNF compatibility with yeast protein engineering and secretion platforms.