Synthetic Binding Proteins Research Articles

A sweet protein monellin as a non-antibody scaffold for synthetic binding proteins.

Synthetic binding proteins that have the ability to bind with molecules can be generated using various protein domains as non-antibody scaffolds. These designer proteins have been used widely in research studies, as their properties overcome the disadvantages of using antibodies. Here, we describe the first application of a phage display to generate synthetic binding proteins using a sweet protein, monellin, as a non-antibody scaffold. Single-chain monellin (scMonellin), in which two polypeptide chains of natural monellin are connected by a short linker, has two loops on one side of the molecule. We constructed phage display libraries of scMonellin, in which the amino acid sequence of the two loops is diversified. To validate the performance of these libraries, we sorted them against the folding mutant of the green fluorescent protein variant (GFPuv) and yeast small ubiquitin-related modifier. We successfully obtained scMonellin variants exhibiting moderate but significant affinities for these target proteins. Crystal structures of one of the GFPuv-binding variants in complex with GFPuv revealed that the two diversified loops were involved in target recognition. scMonellin, therefore, represents a promising non-antibody scaffold in the design and generation of synthetic binding proteins. We termed the scMonellin-derived synthetic binding proteins 'SWEEPins'.

Generation of Protein Inhibitors for Validation of Cancer Drug Targets Identified in Functional Genomic Screens.

Functional genomic screens can identify several proteins as potential targets for drug development in cancer. Typically, these drug targets are validated with pharmacological inhibition using small molecules. Given that chemical inhibitors do not exist for a many of these proteins, several promising candidates often remain unexplored. In this chapter, we describe methods for generating protein-based inhibitors of intracellular targets using phage display. This is a scalable and inexpensive approach that can be applied to several protein targets identified in genetic screens. We describe methods for expression of target proteins, construction of phage-display libraries and selection of binding proteins. These synthetic binding proteins can block natural protein interactions within the cancer cell and act as inhibitors. Protein inhibitors have utility in validation of drug targets and can also guide small-molecule drug development.

Abstract PO-064: Inhibition of RAS signaling and tumorigenesis through novel targeting novel vulnerabilities

Abstract RAS GTPases are important mediators of oncogenesis with nearly 20% of human tumors harboring mutant RAS proteins. However, pharmacological inhibition of RAS has proven challenging. We have employed Monobody technology to discover novel vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies. In contrast to antibodies, Monobodies are fully functional in the reducing environment of the cytoplasm and thus are particularly suitable as genetically encoded “tool biologics”. We previously developed the NS1 Monobody that inhibited RAS by targeting the α4-α5 allosteric lobe to prevent RAS self-association and nanoclustering, and NS1 has become a widely used tool in the RAS research community. Following on this success, we sought to identify additional vulnerabilities in RAS. Based on our discovery that nucleotide-free RAS (apoRAS) inhibits PIK3C2B function, we assessed the feasibility of selectively targeting this state of RAS as an approach to inhibit RAS. Here, we have developed several Monobodies and extensively characterized one of them, R15. Although NS1 was agnostic to the nucleotide state of RAS, R15 bound exclusively to the apo state of all three RAS isoforms but did not interact with nucleotide-loaded RAS. When expressed in cells, R15 inhibited the signaling and transforming activity of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., “fast exchange mutants”), such as G13D and Q61L, but was ineffective against slow exchange mutants such as G12V. Surprisingly, R15 also bound and inhibited RAS(G12D), the most common oncogenic KRAS mutation in pancreatic cancer. Biochemical studies demonstrated that R15 captured RAS from cell lysates in the nucleotide-free state, suggesting that R15 traps apoRAS and prevents nucleotide reloading. Disruption of SOS binding by mutation of D69N further enhanced R15 binding to these fast exchange RAS mutants. Finally, inducible expression of R15 selectively inhibited the tumor forming capacity of tumor lines and patient derived xenografts driven by fast exchange RAS mutants, including RAS(G12D), but not RAS(G12V). Thus, in contrast to conventional wisdom, our approach has established a new opportunity to selectively inhibit the tumorigenic activity of certain RAS mutants by targeting the apo state of RAS with drug-like molecules. Citation Format: Imran Khan, Akiko Koide, Mariyam Zuberi, Eric Denbaum, Gayatari Ketavar, Kai-Wen Teng, Matthew Rhett, Russell Sepncer-Smith, Ernest R. Camp, Shohei Koide, John O’Bryan. Inhibition of RAS signaling and tumorigenesis through novel targeting novel vulnerabilities [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-064.

Abstract B01: Inhibition of RAS signaling and tumorigenesis through targeting vulnerabilities in RAS biochemistry

Abstract RAS GTPases are important mediators of oncogenesis in humans. However, pharmacologic inhibition of RAS has proved challenging. We have taken a novel approach to discover vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies but are insensitive to the redox potential of their environment. We have developed a panel of monobodies that target distinct vulnerabilities in RAS. We recently described the activity of the NS1 monobody at inhibiting RAS signaling. NS1 binds to the α4-α5 allosteric lobe of RAS to prevent RAS dimerization and nanoclustering. When introduced into cells as a genetically encoded reagent, NS1 inhibits RAS signaling and oncogenic transformation in vitro through blocking the ability of RAS to self-associate and stimulate the dimerization and activation of RAF. Using a chemically regulated NS1 expression system, we demonstrate that targeting the α4-α5 dimerization interface with NS1 inhibits KRAS-driven tumors in vivo. In addition to NS1, we will discuss our results with monobodies targeting additional aspects of RAS biochemistry. Our results establish the importance of RAS dimerization through the α4-α5 region in mediating RAS signaling and oncogenic transformation of cells both in vitro and in vivo and reveal additional vulnerabilities in RAS that may be targeted to inhibit RAS-driven tumors. Citation Format: Imran Khan, Russell Spencer-Smith, Kevin Teng, Akiko Koide, Shohei Koide, John P. O'Bryan. Inhibition of RAS signaling and tumorigenesis through targeting vulnerabilities in RAS biochemistry [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B01.

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.

Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic.

Manipulation of the ubiquitin-proteasome pathway to achieve targeted silencing of cellular proteins has emerged as a reliable and customizable strategy for remodeling the mammalian proteome. One such approach involves engineering bifunctional proteins called ubiquibodies that are comprised of a synthetic binding protein fused to an E3 ubiquitin ligase, thus enabling post-translational ubiquitination and degradation of a target protein independent of its function. Here, we have designed a panel of new ubiquibodies based on E3 ubiquitin ligase mimics from bacterial pathogens that are capable of effectively interfacing with the mammalian proteasomal degradation machinery for selective removal of proteins of interest. One of these, the Shigella flexneri effector protein IpaH9.8 fused to a fibronectin type III (FN3) monobody that specifically recognizes green fluorescent protein (GFP), was observed to potently eliminate GFP and its spectral derivatives as well as 15 different FP-tagged mammalian proteins that varied in size (27–179 kDa) and subcellular localization (cytoplasm, nucleus, membrane-associated, and transmembrane). To demonstrate therapeutically relevant delivery of ubiquibodies, we leveraged a bioinspired molecular assembly method whereby synthetic mRNA encoding the GFP-specific ubiquibody was coassembled with poly A binding proteins and packaged into nanosized complexes using biocompatible, structurally defined polypolypeptides bearing cationic amine side groups. The resulting nanoplexes delivered ubiquibody mRNA in a manner that caused efficient target depletion in cultured mammalian cells stably expressing GFP as well as in transgenic mice expressing GFP ubiquitously. Overall, our results suggest that IpaH9.8-based ubiquibodies are a highly modular proteome editing technology with the potential for pharmacologically modulating disease-causing proteins.

A survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins

There is an urgent need for affinity reagents that target phospho-modified sites on individual proteins; however, generating such reagents remains a significant challenge. Here, we describe a genetic selection strategy for routine laboratory isolation of phospho-specific designed ankyrin repeat proteins (DARPins) by linking in vivo affinity capture of a phosphorylated target protein with antibiotic resistance of Escherichia coli cells. The assay is validated using an existing panel of DARPins that selectively bind the nonphosphorylated (inactive) form of extracellular signal-regulated kinase 2 (ERK2) or its doubly phosphorylated (active) form (pERK2). We then use the selection to affinity-mature a phospho-specific DARPin without compromising its selectivity for pERK2 over ERK2 and to reprogram the substrate specificity of the same DARPin towards non-cognate ERK2. Collectively, these results establish our genetic selection as a useful and potentially generalizable protein engineering tool for studying phospho-specific binding proteins and customizing their affinity and selectivity.

In Silico Engineering of Synthetic Binding Proteins from Random Amino Acid Sequences

SummarySynthetic proteins with high affinity and selectivity for a protein target can be used as research tools, biomarkers, and pharmacological agents, but few methods exist to design such proteins de novo. To this end, the In-Silico Protein Synthesizer (InSiPS) was developed to design synthetic binding proteins (SBPs) that bind pre-determined targets while minimizing off-target interactions. InSiPS is a genetic algorithm that refines a pool of random sequences over hundreds of generations of mutation and selection to produce SBPs with pre-specified binding characteristics. As a proof of concept, we design SBPs against three yeast proteins and demonstrate binding and functional inhibition of two of three targets in vivo. Peptide SPOT arrays confirm binding sites, and a permutation array demonstrates target specificity. Our foundational approach will support the field of de novo design of small binding polypeptide motifs and has robust applicability while offering potential advantages over the limited number of techniques currently available.

Biosensors and nanobiosensors for rapid point of care measurement of biomarker proteins

The current gold standard for protein biomarker measurement is the ELISA, which shows good sensitivity and specificity but is typically quite slow and requires expensive and complex equipment. For applications such as early cancer diagnosis, this may limit access, whilst for critical conditions such as acute kidney injury (AKI), sepsis, stroke then rapid measurement has the potential to drive the clinical response. Other applications such as immediate confirmation of bacterial Vs viral infection to inform antibiotic prescribing, also require a fast analysis. Biosensors offer the prospect of reagent-less, processing-free measurements at the patient's bedside. We have fabricated electrochemical immunosensors against a wide range of medical analytes; such sensor operate by virtue of an immobilized bioreceptor, typically an antibody or antibody fragment, or more recently a synthetic binding protein (Affimer) onto an electrode surface. Sensor readout after exposure to the biomarker, in blood, urine or other bio-fluid is then by electrochemical impedance measurement. We have also developed lanthanide ion doped nanoparticles, typically 20–40 nm diameter which are surface-functionalized with antibodies or Affimers. Here, readout after biomarker presentation is via alteration of luminescence intensity, either quenching or enhancement. Also since the nanobiosensors are in suspension and the emission profile can be tuned, then real time measurement, e.g. of the AKI biomarker NGAL in urine should be possible. With all of these devices, the surface nanostructure is absolutely key to performance and this will be discussed.

Facile target validation in an animal model with intracellularly expressed monobodies.

Rapidly determining the biological effect of perturbing a site within a potential drug target could guide drug discovery efforts but it remains challenging. Here, we describe a facile target validation approach that exploits monobodies, small synthetic binding proteins that can be fully functionally expressed in cells. We developed a potent and selective monobody to WDR5, a core component of the Mixed Lineage Leukemia (MLL) methyltransferase complex. The monobody bound to the MLL-interaction site of WDR5, the same site for small molecule inhibitors whose efficacy has been demonstrated in cells but not in animals. As a genetically encoded reagent, the monobody inhibited proliferation of an MLL-AF9 cell line in vitro, suppressed its leukemogenesis and conferred survival benefit in an in vivo mouse leukemia model. The capacity of this approach to readily bridge biochemical, structural, cellular characterization and tests in animal models may accelerate discovery and validation of druggable sites.

Abstract 452: Direct delivery of a synthetic ATP binding protein reduces ATP and induces apoptosis in Hela cells

Abstract Introduction: Reprogramming of cellular metabolism is a hallmark characteristic of cancer cells. As opposed to normal cells, which generate most of their adenine triphosphate (ATP) through oxidative phosphorylation, cancer cells generate ATP less efficiently by aerobic glycolysis (Warburg effect). In addition to deciphering metabolic switches, there is growing interest in understanding the multifaceted role ATP plays in cancer development, progression and chemotherapeutic resistance. One approach to untangling these roles is to significantly reduce intracellular ATP and investigate the cellular response. With that, we designed and created a man-made protein that chelates ATP with high specificity and affinity. This synthetic protein, called DX, has been genetically expressed in a living organism, Escherichia coli, where it reduced intracellular ATP to below the levels of detection. Hypothesis: Delivery of the DX protein into HeLa cancer cells will reduce intracellular bioavailable ATP affecting energy metabolism, viability and drug susceptibility. Methods: A cationic lipid mixture was complexed with active, purified DX protein to generate a DX/lipid complex. The integrity of the complexed protein was verified by western blot analysis. HeLa cells were incubated with the DX/lipid complex to allow efficient delivery of DX into the cytoplasm of the HeLa cells. The impact of DX on cell viability was determined using a tetrazolium-based colorimetric cell viability assay and a caspase 3/7 assay. To correlate phenotypic/viability change with DX activity, bioavailable ATP levels were measured at specific time points following DX delivery. To establish whether DX impacts drug export, the retention of calcein-AM, a substrate of the ATP-dependent p-glycoprotein pump, was measured post DX-delivery. Results: Western blot analysis confirmed the stability of DX prior to- and post- delivery to HeLa cells. In a time- and dose- dependent manner, DX negatively impacted cell growth and induced cell death via apoptosis, at a time concomitant with a decrease in bioavailable ATP. Significantly, HeLa cells treated with DX retained calcein-AM, suggesting reduced p-glycoprotein activity. Conclusion: Advances in protein engineering have made it possible to create artificial proteins with specific functions. In addition to direct clinical applications, synthetic proteins are powerful tools that have the potential to reveal something new about biology. Direct delivery of a synthetic ATP-binding protein potentiated apoptosis in HeLa cells contemporaneous with reduced intracellular ATP. Having established DX activity in a living system, future studies will examine the cancer cell metabolome in response to significant ATP stress. Finally, a reduction in p-glycoprotein activity in DX-treated HeLa cells advocates examination of concurrent DX/chemotherapeutic treatment, principally in drug resistant scenarios. Citation Format: Selina M. Martinez, Shanika Kingston, Arlene Ngor, John C. Chaput, Nagaraj J. Vinay, Shaleen B. Korch. Direct delivery of a synthetic ATP binding protein reduces ATP and induces apoptosis in Hela cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 452.

Monobody-Mediated Alteration of Lipase Substrate Specificity.

Controlling the catalytic properties of enzymes remain an important challenge in chemistry and biotechnology. We have recently established a strategy for altering enzyme specificity in which the addition of proxy monobodies, synthetic binding proteins, modulates the specificity of an otherwise unmodified enzyme. Here, in order to examine its broader applicability, we employed the strategy on Candida rugosa lipase 1 (CRL1), an enzyme with a tunnel-like substrate binding site. We successfully identified proxy monobodies that restricted the substrate specificity of CRL1 toward short-chain fatty acids. The successes with this enzyme system and a β-galactosidase used in the previous work suggest that our strategy can be applied to diverse enzymes with distinct architectures of substrate binding sites.

Monobodies as possible next-generation protein therapeutics - a perspective.

SummaryOver the past two decades, hundreds of new somatic mutations have been identified in tumours, and a few dozen novel cancer therapeutics that selectively target these mutated oncoproteins have entered clinical practice. This development has resulted in clinical breakthroughs for a few tumour types, but more commonly patients' overall survival has not improved because of the development of drug resistance. Furthermore, only a very limited number of oncoproteins, largely protein kinases, are successfully targeted, whereas most non-kinase oncoproteins inside cancer cells remain untargeted. Engineered small protein inhibitors offer great promise in targeting a larger variety of oncoproteins with better efficacy and higher selectivity. In this article, I focus on a promising class of synthetic binding proteins, termed monobodies, that we have shown to inhibit previously untargetable protein-protein interactions in different oncoproteins. I will discuss the great promise alongside the technical challenges inherent in converting monobodies from potent pre-clinical target validation tools to next-generation protein-based therapeutics.

Rigidly connected multispecific artificial binders with adjustable geometries

Multivalent binding proteins can gain biological activities beyond what is inherent in the individual binders, by bringing together different target molecules, restricting their conformational flexibility or changing their subcellular localization. In this study, we demonstrate a method to build up rigid multivalent and multispecific scaffolds by exploiting the modular nature of a repeat protein scaffold and avoiding flexible linkers. We use DARPins (Designed Ankyrin Repeat Proteins), synthetic binding proteins based on the Ankyrin-repeat protein scaffold, as binding units. Their ease of in vitro selection, high production yield and stability make them ideal specificity-conferring building blocks for the design of more complex constructs. C- and N-terminal DARPin capping repeats were re-designed to be joined by a shared helix in such a way that rigid connector modules are formed. This allows us to join two or more DARPins in predefined geometries without compromising their binding affinities and specificities. Nine connector modules with distinct geometries were designed; for eight of these we were able to confirm the structure by X-ray crystallography, while only one did not crystallize. The bispecific constructs were all able to bind both target proteins simultaneously.

Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody–SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells.

Monobodies and other synthetic binding proteins for expanding protein science.

Synthetic binding proteins are constructed using nonantibody molecular scaffolds. Over the last two decades, in‐depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function, often leading to new mechanistic insights. We propose that synthetic proteins will become powerful standard tools in diverse areas of protein science, biotechnology and medicine.

Inhibition of RAS function through targeting an allosteric regulatory site.

RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-β6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-β6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

Size-dependent protein segregation at membrane interfaces.

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane protein organization, such as E-cadherin enrichment in epithelial junctional complexes and CD45 exclusion from the signaling foci of immunological synapses. To isolate the role of protein size in these processes, we reconstituted membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between binding and non-binding proteins can dramatically alter their organization at membrane interfaces in the absence of active contributions from the cytoskeleton, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally-driven membrane height fluctuations that transiently limit access to the interface. This simple, sensitive, and highly effective means of passively segregating proteins has implications for signaling at cell-cell junctions and protein sorting at intracellular contact points between membrane-bound organelles.

ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation.

Prdm14 is a sequence-specific transcriptional regulator of embryonic stem cell (ESC) pluripotency and primordial germ cell (PGC) formation. It exerts its function, at least in part, through repressing genes associated with epigenetic modification and cell differentiation. Here, we show that this repressive function is mediated through an ETO-family co-repressor Mtgr1, which tightly binds to the pre-SET/SET domains of Prdm14 and co-occupies its genomic targets in mouse ESCs. We generated two monobodies, synthetic binding proteins, targeting the Prdm14 SET domain and demonstrate their utility, respectively, in facilitating crystallization and structure determination of the Prdm14-Mtgr1 complex, or as genetically encoded inhibitor of the Prdm14-Mtgr1 interaction. Structure-guided point mutants and the monobody abrogated the Prdm14-Mtgr1 association and disrupted Prdm14's function in mESC gene expression and PGC formation in vitro. Altogether, our work uncovers the molecular mechanism underlying Prdm14-mediated repression and provides renewable reagents for studying and controlling Prdm14 functions.

Monobody-mediated alteration of enzyme specificity

Current methods for engineering enzymes modify enzymes themselves and require a detailed mechanistic understanding or a high-throughput assay. Here, we describe a new approach where catalytic properties are modulated with synthetic binding proteins, termed monobodies, directed to an unmodified enzyme. Using the example of a β-galactosidase from Bacillus circulans, we efficiently identified monobodies that restricted its substrates for its transgalactosylation reaction and selectively enhanced the production of small oligosaccharide prebiotics.