Sarel Fleishman

Unspecific peroxygenases (UPOs) are fungal enzymes that attract significant attention for their ability to perform versatile oxyfunctionalization reactions using H2O2. Unlike other oxygenases, UPOs do not require additional reductive equivalents or electron transfer chains that complicate basic and applied research. Nevertheless, UPOs generally exhibit low to no heterologous production levels and only four UPO structures have been determined to date by crystallography limiting their usefulness and obstructing research. To overcome this bottleneck, we implemented a workflow that applies PROSS stability design to AlphaFold2 model structures of 10 unique and diverse UPOs followed by a signal peptide shuffling to enable heterologous production. Nine UPOs were functionally produced in Pichia pastoris, including the recalcitrant CciUPO and three UPOs derived from oomycetes─the first nonfungal UPOs to be …

Provided herein are Cas9 proteins comprising SEQ ID NO: 1 or a sequence at least 80% identical thereto, wherein: the amino acid residues at positions 765 to 780 are replaced by the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6; the amino acid residues at positions 838 to 853 are replaced by the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10; and/or the amino acid residues at positions 911 to 925 are replaced by the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13. Also provided are Cas9 proteins comprising an HNH domain comprising the amino acid sequence of SEQ ID NO: 14 or a sequence at least 80% identical thereto, wherein: the amino acid residues at positions 1 to 16 are replaced by the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6; the amino acid residues at positions 74 to 89 are replaced by the amino acid …

The development of a highly effective vaccine against the pathogenic blood-stage infection of human malaria will require a delivery platform that can induce an antibody response of both maximal quantity and functional quality. One strategy to achieve this includes presenting antigens to the immune system on virus-like particles (VLPs). Here we sought to improve the design and delivery of the blood-stage Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) antigen, which is currently in a Phase 2 clinical trial as a full-length soluble protein-in-adjuvant vaccine candidate called RH5.1/Matrix-M™. We identify disordered regions of the full-length RH5 molecule induce non-growth inhibitory antibodies in human vaccinees, and a re-engineered and stabilized immunogen that includes just the alpha-helical core of RH5 induces a qualitatively superior growth-inhibitory antibody response in rats vaccinated with this protein formulated in Matrix-M™ adjuvant. In parallel, bioconjugation of this new immunogen, termed ″RH5.2″, to hepatitis B surface antigen VLPs using the ″plug-and-display″ SpyTag-SpyCatcher platform technology also enabled superior quantitative antibody immunogenicity over soluble antigen/adjuvant in vaccinated mice and rats. These studies identify a new blood-stage malaria vaccine candidate that may improve upon the current leading soluble protein vaccine candidate RH5.1/Matrix-M™. The RH5.2-VLP/Matrix-M™ vaccine candidate is now under evaluation in Phase 1a/b clinical trials.

Almost all attempts to date at gene therapy approaches for monogenetic disease have used the amino acid sequences of the natural protein. In the current study, we use a designed, thermostable form of glucocerebrosidase (GCase), the enzyme defective in Gaucher disease (GD), to attempt to alleviate neurological symptoms in a GD mouse that models type 3 disease, ie the chronic neuronopathic juvenile subtype. Upon injection of an AAVrh10 (adeno-associated virus, serotype rh10) vector containing the designed GCase (dGCase) into the left lateral ventricle of Gba-/-; Gbatg mice, a significant improvement in body weight and life-span was observed, compared to injection of the same mouse with the wild type enzyme (wtGCase). Moreover, a reduction in levels of glucosylceramide (GlcCer), and an increase in levels of GCase activity were seen in the right hemisphere of Gba-/-; Gbatg mice, concomitantly with a significant improvement in motor function, reduction of neuroinflammation and a reduction in mRNA levels of various genes shown previously to be elevated in the brain of mouse models of neurological forms of GD. Together, these data pave the way for the possible use of modified proteins in gene therapy for lysosomal storage diseases and other monogenetic disorders.

Conventional methods for humanizing animal-derived antibodies involve grafting their complementarity-determining regions onto homologous human framework regions. However, this process can substantially lower antibody stability and antigen-binding affinity, and requires iterative mutational fine-tuning to recover the original antibody properties. Here we report a computational method for the systematic grafting of animal complementarity-determining regions onto thousands of human frameworks. The method, which we named CUMAb (for computational human antibody design; available at http://CUMAb.weizmann.ac.il), starts from an experimental or model antibody structure and uses Rosetta atomistic simulations to select designs by energy and structural integrity. CUMAb-designed humanized versions of five antibodies exhibited similar affinities to those of the parental animal antibodies, with some designs …

The field of protein design has made remarkable progress over the past decade. Historically, the low reliability of purely structure-based design methods limited their application, but recent strategies that combine structure-based and sequence-based calculations, as well as machine learning tools, have dramatically improved protein engineering and design. In this Review, we discuss how these methods have enabled the design of increasingly complex structures and therapeutically relevant activities. Additionally, protein optimization methods have improved the stability and activity of complex eukaryotic proteins. Thanks to their increased reliability, computational design methods have been applied to improve therapeutics and enzymes for green chemistry and have generated vaccine antigens, antivirals and drug-delivery nano-vehicles. Moreover, the high success of design methods reflects an increased …

Protein degradation is one of the essential mechanisms that enables reshaping of the proteome landscape in response to various stimuli. The largest E3 ubiquitin ligase family that targets proteins to degradation by catalyzing ubiquitination is the cullin–RING ligases (CRLs). Many of the proteins that are regulated by CRLs are central to tumorigenesis and tumor progression, and dysregulation of the CRL family is frequently associated with cancer. The CRL family comprises ∼300 complexes, all of which are regulated by the COP9 signalosome complex (CSN). Therefore, CSN is considered an attractive target for therapeutic intervention. Research efforts for targeted CSN inhibition have been directed towards inhibition of the complex enzymatic subunit, CSN5. Here, we have taken a fresh approach focusing on CSNAP, the smallest CSN subunit. Our results show that the C-terminal region of CSNAP is tightly packed …

Several methods have been developed to explore interactions among water-soluble proteins or regions of proteins. However, techniques to target transmembrane domains (TMDs) have not been examined thoroughly despite their importance. Here, we developed a computational approach to design sequences that specifically modulate protein–protein interactions in the membrane. To illustrate this method, we demonstrated that BclxL can interact with other members of the B cell lymphoma 2 (Bcl2) family through the TMD and that these interactions are required for BclxL control of cell death. Next, we designed sequences that specifically recognize and sequester the TMD of BclxL. Hence, we were able to prevent BclxL intramembrane interactions and cancel its antiapoptotic effect. These results advance our understanding of protein–protein interactions in membranes and provide a means to modulate them …

Golden Gate (GG) assembly is an effective tool for the seamless assembly of genes from DNA fragments. Its use for generating combinatorial mutagenic libraries is much less developed. We describe a new method called GGAssembler for designing gene libraries that encode combinatorial mutations through GG gene assembly. GGAssembler uses a graph-theoretical approach to find the most cost-effective way to assemble diversity from DNA fragments. We demonstrate the accuracy, efficiency, low bias, and cost-effectiveness of GGAssembler by encoding hundreds of thousands of variants of camelid single-domain antibodies. We envision that GGAssembler will advance protein science and engineering by enabling the construction of diverse protein libraries with precise control over mutations. Source code is freely available at: https://github. com/Fleishman-Lab/GGAssembler.

Acid‐β‐glucosidase (GCase, EC3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme®) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step toward developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability‐design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild‐type human GCase …

The generation of enantiodivergent biocatalysts for C–H oxyfunctionalizations is ever more important in modern synthetic chemistry. Here, we have applied the FuncLib algorithm based on phylogenetic and Rosetta calculations to design a diverse repertoire of active, stable, and enantiodivergent fungal peroxygenases. 24 designs, each carrying 4–5 mutations in the catalytic core, were expressed functionally in yeast and benchmarked against characteristic model compounds. Several designs were active and stable in a range of temperature and pH, displaying unprecedented enantiodivergence, changing regioselectivity from alkyl to aromatic hydroxylation, and increasing catalytic efficiencies up to 10-fold, with 15-fold improvements in total turnover numbers over the parental enzyme. We find that this dramatic functional divergence stems from beneficial epistasis among the mutations and an extensive …

Golden Gate (GG) assembly is an effective tool for the seamless assembly of genes from DNA fragments. Its use for generating combinatorial mutagenic libraries is much less developed. We describe a new method called GGAssembler for designing gene libraries that encode combinatorial mutations through GG gene assembly. GGAssembler uses a graph-theoretical approach to find the most cost-effective way to assemble diversity from DNA fragments. We demonstrate the accuracy, efficiency, low bias, and cost-effectiveness of GGAssembler by encoding hundreds of thousands of variants of camelid single-domain antibodies. We envision that GGAssembler will advance protein science and engineering by enabling the construction of diverse protein libraries with precise control over mutations. Source code is freely available at: https://github. com/Fleishman-Lab/GGAssembler.

Provided herein is a family of programmable de novo designed transmembranal protein domain polypeptides, and uses thereof in the production of engineered chimeric antigen receptor T (CAR T) cells, which are used, for example, to fight cancer.

The design of structurally diverse enzymes is constrained by long-range interactions that are necessary for accurate folding. We introduce an atomistic and machine learning strategy for the combinatorial assembly and design of enzymes (CADENZ) to design fragments that combine with one another to generate diverse, low-energy structures with stable catalytic constellations. We applied CADENZ to endoxylanases and used activity-based protein profiling to recover thousands of structurally diverse enzymes. Functional designs exhibit high active-site preorganization and more stable and compact packing outside the active site. Implementing these lessons into CADENZ led to a 10-fold improved hit rate and more than 10,000 recovered enzymes. This design-test-learn loop can be applied, in principle, to any modular protein family, yielding huge diversity and general lessons on protein design principles.

A genetically modified human beta-glucocerebrosidase (GCase) is disclosed. The genetically modified GCase comprising an amino acid sequence at least 85% identical to SEQ ID NO: 2; and comprising mutations at coordinates L34P, K224N/G, T369E and N370D, where the coordinates correspond to said SEQ ID NO: 2; and capable of catalyzing hydrolysis of a glycolipid glucosylceramide (GlcCer). Pharmaceutical compositions comprising the genetically modified GCase and therapeutic methods of using same are also disclosed.

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type …

Until now, membrane-protein stabilization has relied on iterations of mutations and screening. We now validate a one-step algorithm, mPROSS, for stabilizing membrane proteins directly from an AlphaFold2 model structure. Applied to the lipid-generating enzyme, ceramide synthase, 37 designed mutations lead to a more stable form of human CerS2. Together with molecular dynamics simulations, we propose a pathway by which substrates might be delivered to the ceramide synthases.

Controlled degradation of proteins is necessary for ensuring their abundance and sustaining a healthy and accurately functioning proteome. One of the degradation routes involves the uncapped 20S proteasome, which cleaves proteins with a partially unfolded region, including those that are damaged or contain intrinsically disordered regions. This degradation route is tightly controlled by a recently discovered family of proteins named Catalytic Core Regulators (CCRs). Here, we show that CCRs function through an allosteric mechanism, coupling the physical binding of the PSMB4 β-subunit with attenuation of the complex’s three proteolytic activities. In addition, by dissecting the structural properties that are required for CCR-like function, we could recapitulate this activity using a designed protein that is half the size of natural CCRs. These data uncover an allosteric path that does not involve the proteasome’s …

GuidelinesPlease note that protocols with Q5® High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.

Some protein binding pairs exhibit extreme specificities that functionally insulate them from homologs. Such pairs evolve mostly by accumulating single-point mutations, and mutants are selected if their affinity exceeds the threshold required for function 1–4. Thus, homologous and high-specificity binding pairs bring to light an evolutionary conundrum: how does a new specificity evolve while maintaining the required affinity in each intermediate 5, 6? Until now, a fully functional single-mutation path that connects two orthogonal pairs has only been described where the pairs were mutationally close thus enabling experimental enumeration of all intermediates 2. We present an atomistic and graph-theoretical framework for discovering low molecular strain single-mutation paths that connect two extant pairs, enabling enumeration beyond experimental capability. We apply it to two orthogonal bacterial colicin …