Antibody fragments functionalized with non-canonical amino acids preserving structure and functionality - A door opener for new biological and therapeutic applications.

Functionalization of proteins by incorporating reactive non-canonical amino acids (ncAAs) has been widely applied for numerous biological and therapeutic applications. The requirement not to lose the intrinsic properties of these proteins is often underestimated and not considered. Main purpose of this study was to answer the question whether functionalization via residue-specific incorporation of the ncAA N6-[(2-Azidoethoxy) carbonyl]-l-lysine (Azk) influences the properties of the anti-tumor-necrosis-factor-α-Fab (FTN2). Therefore, FTN2Azk variants with different Azk incorporation sites were designed and amber codon suppression was used for production. The functionalized FTN2Azk variants were efficiently produced in fed-batch like µ-bioreactor cultivations in the periplasm of E. coli displaying correct structure and antigen binding affinities comparable to those of wild-type FTN2. Our FTN2Azk variants with reactive handles for diverse conjugates enable tracking of recombinant protein in the production cell, pharmacological studies and translation into new pharmaceutical applications.

Lectin-anticancer peptide fusion demonstrates a significant cancer-cell-selective cytotoxic effect and inspires the production of "clickable" anticancer peptide in Escherichia coli.

Targeted killing of tumor cells while protecting healthy cells is the pressing priority in cancer treatment. Lectins that target a specific glycan marker abundant in cancer cells can be valuable new tools for selective cancer cell killing. The lectin Shiga-like toxin 1 B subunit (Stx1B) is an example that specifically binds globotriaosylceramide (CD77 or Gb3), which is overexpressed in certain cancers. In this study, a human lactoferricin-derived synthetic retro di-peptide R-DIM-P-LF11-215 with antitumor efficacy was fused to the lectin Stx1B to selectively target and kill Gb3+ cancer cells. We produced lectin-peptide fusion proteins in Escherichia coli, isolated them by Gb3-affinity chromatography, and assessed their ability to selectively kill Gb3+ cancer cells in a Calcein AM assay. Furthermore, to expand the applications of R-DIM-P-LF11-215 in developing therapeutic bioconjugates, we labeled R-DIM-P-LF11-215 with the unique reactive non-canonical amino acid Nε -((2-azidoethoxy)carbonyl)-L-lysine (AzK) at a selected position by amber stop codon suppression. The R-DIM-P-LF11-215 20AzK and the unlabeled R-DIM-P-LF11-215 parent peptide were produced as GST-fusion proteins for soluble expression in E. coli for the first time. We purified both variants by size-exclusion chromatography and analyzed their peptide masses. Finally, a cyanin 3 fluorophore was covalently conjugated to R-DIM-P-LF11-215 20AzK by strain-promoted alkyne-azide cycloaddition. Our results showed that the recombinant lectin-peptide fusion R-DIM-P-LF11-215-Stx1B killed >99% Gb3+ HeLa cells while Gb3-negative cells were unaffected. The peptides R-DIM-P-LF11-215 and R-DIM-P-LF11-215 20AzK were produced recombinantly in E. coli in satisfactory amounts and were tested functional by cytotoxicity and cell-binding assays, respectively.

Clickable Shiga Toxin B Subunit for Drug Delivery in Cancer Therapy.

In recent years, receptor-mediated drug delivery has gained major attention in the treatment of cancer. The pathogen-derived Shiga Toxin B subunit (STxB) can be used as a carrier that detects the tumor-associated glycosphingolipid globotriaosylceramide (Gb3) receptors. While drug conjugation via lysine or cysteine offers random drug attachment to carriers, click chemistry has the potential to improve the engineering of delivery systems as the site specificity can eliminate interference with the active binding site of tumor ligands. We present the production of recombinant STxB in its wild-type (STxBwt) version or incorporating the noncanonical amino acid azido lysine (STxBAzK). The STxBwt and STxBAzK were manufactured using a growth-decoupled Escherichia coli (E. coli)-based expression strain and analyzed via flow cytometry for Gb3 receptor recognition and specificity on two human colorectal adenocarcinoma cell lines-HT-29 and LS-174-characterized by high and low Gb3 abundance, respectively. Furthermore, STxBAzK was clicked to the antineoplastic agent monomethyl auristatin E (MMAE) and evaluated in cell-killing assays for its ability to deliver the drug to Gb3-expressing tumor cells. The STxBAzK-MMAE conjugate induced uptake and release of the MMAE drug in Gb3-positive tumor cells, reaching 94% of HT-29 cell elimination at 72 h post-treatment and low nanomolar doses while sparing LS-174 cells. STxBAzK is therefore presented as a well-functioning drug carrier, with a possible application in cancer therapy. This research demonstrates the feasibility of lectin carriers used in delivering drugs to tumor cells, with prospects for improved cancer therapy in terms of straightforward drug attachment and effective cancer cell elimination.

Genetic code expansion in E. coli enables production of a functional 'ready-to-click' T cell receptor-specific scFv.

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable VH and VL domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition ('click chemistry'). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a 'ready-to-click' scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

Bidirectional Promoter Libraries Enable the Balanced Co-expression of Two Target Genes in E. coli.

In this chapter, we present a bidirectional promoter library toolbox to evaluate fast and efficiently the optimal conditions for the balanced co-expression of two target genes. As a proof-of-concept, we demonstrate the co-expression of CYP505x and the GroEL/ES complex, which resulted in noticeably elevated enzyme activity with one of the de-novo-designed promoters of the library. The new toolbox offers a straightforward one-pot cloning approach and is highly modular. As such, the method presented here should be of great interest to any gene co-expression study.

A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death.

BACKGROUND: Aberrant glycosylation patterns play a crucial role in the development of cancer cells as they promote tumor growth and aggressiveness. Lectins recognize carbohydrate antigens attached to proteins and lipids on cell surfaces and represent potential tools for application in cancer diagnostics and therapy. Among the emerging cancer therapies, immunotherapy has become a promising treatment modality for various hematological and solid malignancies. Here we present an approach to redirect the immune system into fighting cancer by targeting altered glycans at the surface of malignant cells. We developed a so-called "lectibody", a bispecific construct composed of a lectin linked to an antibody fragment. This lectibody is inspired by bispecific T cell engager (BiTEs) antibodies that recruit cytotoxic T lymphocytes (CTLs) while simultaneously binding to tumor-associated antigens (TAAs) on cancer cells. The tumor-related glycosphingolipid globotriaosylceramide (Gb3) represents the target of this proof-of-concept study. It is recognized with high selectivity by the B-subunit of the pathogen-derived Shiga toxin, presenting opportunities for clinical development. METHODS: The lectibody was realized by conjugating an anti-CD3 single-chain antibody fragment to the B-subunit of Shiga toxin to target Gb3+ cancer cells. The reactive non-canonical amino acid azidolysine (AzK) was inserted at predefined single positions in both proteins. The azido groups were functionalized by bioorthogonal conjugation with individual linkers that facilitated selective coupling via an alternative bioorthogonal click chemistry reaction. In vitro cell-based assays were conducted to evaluate the antitumoral activity of the lectibody. CTLs, Burkitt´s lymphoma-derived cells and colorectal adenocarcinoma cell lines were screened in flow cytometry and cytotoxicity assays for activation and lysis, respectively. RESULTS: This proof-of-concept study demonstrates that the lectibody activates T cells for their cytotoxic signaling, redirecting CTLs´ cytotoxicity in a highly selective manner and resulting in nearly complete tumor cell lysis-up to 93%-of Gb3+ tumor cells in vitro. CONCLUSIONS: This research highlights the potential of lectins in targeting certain tumors, with an opportunity for new cancer treatments. When considering a combinatorial strategy, lectin-based platforms of this type offer the possibility to target glycan epitopes on tumor cells and boost the efficacy of current therapies, providing an additional strategy for tumor eradication and improving patient outcomes.

One, two, many: Strategies to alter the number of carbohydrate binding sites of lectins.

Carbohydrates are more than an energy-storage. They are ubiquitously found on cells and most proteins, where they encode biological information. Lectins bind these carbohydrates and are essential for translating the encoded information into biological functions and processes. Hundreds of lectins are known, and they are found in all domains of life. For half a century, researchers have been preparing variants of lectins in which the binding sites are varied. In this way, the traits of the lectins such as the affinity, avidity and specificity towards their ligands as well as their biological efficacy were changed. These efforts helped to unravel the biological importance of lectins and resulted in improved variants for biotechnological exploitation and potential medical applications. This review gives an overview on the methods for the preparation of artificial lectins and complexes thereof and how reducing or increasing the number of binding sites affects their function.

Fast Protein Modification in the Nanomolar Concentration Range Using an Oxalyl Amide as Latent Thioester.

We show that latent oxalyl thioester surrogates are a powerful means to modify peptides and proteins in highly dilute conditions in purified aqueous media or in mixtures as complex as cell lysates. Designed to be shelf-stable reagents, they can be activated on demand to enable ligation reactions with peptide concentrations as low as a few hundred nM at rates approaching 30 M-1 s-1 .

Engineering cascade biocatalysis in whole cells for bottom-up synthesis of cello-oligosaccharides: flux control over three enzymatic steps enables soluble production.

BACKGROUND: Soluble cello-oligosaccharides (COS, ß-1,4-D-gluco-oligosaccharides with degree of polymerization DP 2-6) have been receiving increased attention in different industrial sectors, from food and feed to cosmetics. Development of large-scale COS applications requires cost-effective technologies for their production. Cascade biocatalysis by the three enzymes sucrose-, cellobiose- and cellodextrin phosphorylase is promising because it enables bottom-up synthesis of COS from expedient substrates such as sucrose and glucose. A whole-cell-derived catalyst that incorporates the required enzyme activities from suitable co-expression would represent an important step towards making the cascade reaction fit for production. Multi-enzyme co-expression to reach distinct activity ratios is challenging in general, but it requires special emphasis for the synthesis of COS. Only a finely tuned balance between formation and elongation of the oligosaccharide precursor cellobiose results in the desired COS. RESULTS: Here, we show the integration of cellodextrin phosphorylase into a cellobiose-producing whole-cell catalyst. We arranged the co-expression cassettes such that their expression levels were upregulated. The most effective strategy involved a custom vector design that placed the coding sequences for cellobiose phosphorylase (CbP), cellodextrin phosphorylase (CdP) and sucrose phosphorylase (ScP) in a tricistron in the given order. The expression of the tricistron was controlled by the strong T7lacO promoter and strong ribosome binding sites (RBS) for each open reading frame. The resulting whole-cell catalyst achieved a recombinant protein yield of 46% of total intracellular protein in an optimal ScP:CbP:CdP activity ratio of 10:2.9:0.6, yielding an overall activity of 315 U/g dry cell mass. We demonstrated that bioconversion catalyzed by a semi-permeabilized whole-cell catalyst achieved an industrial relevant COS product titer of 125 g/L and a space-time yield of 20 g/L/h. With CbP as the cellobiose providing enzyme, flux into higher oligosaccharides (DP ≥ 6) was prevented and no insoluble products were formed after 6 h of conversion. CONCLUSIONS: A whole-cell catalyst for COS biosynthesis was developed. The coordinated co-expression of the three biosynthesis enzymes balanced the activities of the individual enzymes such that COS production was maximized. With the flux control set to minimize the share of insolubles in the product, the whole-cell synthesis shows a performance with respect to yield, productivity, product concentration and quality that is promising for industrial production.

Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters.

Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in Thermoanaerobacter thermohydrosulfuricus lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met-142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in significantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These findings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering.

Engineering the Ligand Specificity of the Human Galectin-1 by Incorporation of Tryptophan Analogues.

Galectin-1 is a ß-galactoside-binding lectin with manifold biological functions. A single tryptophan residue (W68) in its carbohydrate binding site plays a major role in ligand binding and is highly conserved among galectins. To fine tune galectin-1 specificity, we introduced several non-canonical tryptophan analogues at this position of human galectin-1 and analyzed the resulting variants using glycan microarrays. Two variants containing 7-azatryptophan and 7-fluorotryptophan showed a reduced affinity for 3'-sulfated oligosaccharides. Their interaction with different ligands was further analyzed by fluorescence polarization competition assay. Using molecular modeling we provide structural clues that the change in affinities comes from modulated interactions and solvation patterns. Thus, we show that the introduction of subtle atomic mutations in the ligand binding site of galectin-1 is an attractive approach for fine-tuning its interactions with different ligands.

Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli.

The site-specific incorporation of noncanonical amino acids (ncAAs) into proteins by amber stop codon suppression has become a routine method in academic laboratories. This approach requires an amber suppressor tRNACUA to read the amber codon and an aminoacyl-tRNA synthetase to charge the tRNACUA with the ncAA. However, a major drawback is the low yield of the mutant protein in comparison to the wild type. This effect primarily results from the competition of release factor 1 with the charged suppressor tRNACUA for the amber codon at the A-site of the ribosome. A number of laboratories have attempted to improve the incorporation efficiency of ncAAs with moderate results. We aimed at increasing the efficiency to produce high yields of ncAA-functionalized proteins in a scalable setting for industrial application. To do this, we inserted an ncAA into the enhanced green fluorescent protein and an antibody mimetic molecule using an industrial E. coli strain, which produces recombinant proteins independent of cell growth. The controlled decoupling of recombinant protein production from cell growth considerably increased the incorporation of the ncAA, producing substantially higher protein yields versus the reference E. coli strain BL21(DE3). The target proteins were expressed at high levels, and the ncAA was efficiently incorporated with excellent fidelity while the protein function was preserved.

Plasmid Design for Tunable Two-Enzyme Co-Expression Promotes Whole-Cell Production of Cellobiose.

Catalyst development for biochemical cascade reactions often follows a "whole-cell-approach" in which a single microbial cell is made to express all required enzyme activities. Although attractive in principle, the approach can encounter limitations when efficient overall flux necessitates precise balancing between activities. This study shows an effective integration of major design strategies from synthetic biology to a coherent development of plasmid vectors, enabling tunable two-enzyme co-expression in E. coli, for whole-cell-production of cellobiose. An efficient transformation of sucrose and glucose into cellobiose by a parallel (countercurrent) cascade of disaccharide phosphorylases requires the enzyme co-expression to cope with large differences in specific activity of cellobiose phosphorylase (14 U mg-1 ) and sucrose phosphorylase (122 U mg-1 ). Mono- and bicistronic co-expression strategies controlling transcription, transcription-translation coupling or plasmid replication are analyzed for effect on activity and stable producibility of the whole-cell-catalyst. A key role of bom (basis of mobility) for plasmid stability dependent on the ori is reported and the importance of RBS (ribosome binding site) strength is demonstrated. Whole cell catalysts show high specific rates (460 µmol cellobiose min-1  g-1 dry cells) and performance metrics (30 g L-1 ; ∼82% yield; 3.8 g L-1 h-1 overall productivity) promising for cellobiose production.

Enzymes revolutionize the bioproduction of value-added compounds: From enzyme discovery to special applications.

Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty- and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

'Clickable lectins': bioorthogonal reactive handles facilitate the directed conjugation of lectins in a modular fashion.

Lectins are carbohydrate-binding proteins with specificity for their target ligands. They play diverse roles in cellular recognition and signalling processes, as well as in infections and cancer metastasis. Owing to their specificity, lectins find application in biotechnology and medicine, e.g. for blood group typing, purification of glycoproteins or lipids and as markers that target cancer cells. For some applications, lectins are immobilized on a solid support, or they are conjugated with other molecules. Classical protein conjugation reactions at nucleophilic amino acids such as cysteine or lysine are often non-selective, and the site of conjugation is difficult to pre-define. Random conjugation, however, can interfere with protein function. Therefore, we sought to equip lectins with a unique reactive handle, which can be conjugated with other molecules in a pre-defined manner. We site-specifically introduced non-canonical amino acids carrying bioorthogonal reactive groups into several lectins. As a proof of principle, we conjugated these 'clickable lectins' with small molecules. Furthermore, we conjugated lectins with different ligand specificities with one another to produce superlectins. The 'clickable lectins' might be useful for any process where lectins shall be conjugated with another module in a selective, pre-defined and site-specific manner.

Substituting the catalytic proline of 4-oxalocrotonate tautomerase with non-canonical analogues reveals a finely tuned catalytic system.

The enzyme 4-oxalocrotonate tautomerase shows remarkable catalytic versatility due to the secondary amine of its N-terminal proline moiety. In this work, we incorporated a range of proline analogues into the enzyme and examined the effects on structure and activity. While the structure of the enzyme remained unperturbed, its promiscuous Michael-type activity was severely affected. This finding demonstrates how atomic changes in a biocatalytic system can abolish its activity. Our work provides a toolbox for successful generation of enzyme variants with non-canonical catalytic proline analogues.

A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry.

The site-specific incorporation of non-canonical amino acids (ncAAs) at amber codons requires an aminoacyl-tRNA synthetase and a cognate amber suppressor tRNA (tRNACUA ). The archaeal tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei have been extensively engineered to accept a versatile set of ncAAs. The PylRS/tRNACUA pair from the bacterium Desulfitobacterium hafniense is functional in Escherichia coli, however, variants of this PylRS have not been reported yet. In this study, the authors describe a bacterial PylRS from Desulfitobacterium hafniense, which the authors engineered for the reactive ncAA para-azido-l-phenylalanine (DhAzFRS) using a semi-rational approach. DhAzFRS preferred para-azido-l-phenylalanine to the canonical l-phenylalanine as the substrate. In addition, the authors demonstrate the functionality in E. coli of a hybrid DhAzFRS carrying the first 190 N-terminal amino acids of the Methanosarcina mazei PylRS. These results suggest that bacterial and archaeal PylRSs can be "mixed and matched" to tune their substrate specificity.

Molecular cloning, expression, and characterization of acyltransferase from Pseudomonas protegens.

The formation of C-C bonds by using CoA independent acyltransferases may have significant impact for novel methods for biotechnology. We report the identification of Pseudomonas strains with CoA-independent acyltransferase activity as well as the heterologous expression of the enzyme in E. coli. The cloning strategies and selected expression studies are discussed. The recombinant acyltransferases were characterized with regard to thermal and storage stability, pH,- and co-solvent tolerance. Moreover, the impact of bivalent metals, inhibitors, and other additives was tested. Careful selection of expression and working conditions led to obtain recombinant acyltransferase form Pseudomonas protegens with up to 11 U mL-1 activity.

Effect of Noncanonical Amino Acids on Protein-Carbohydrate Interactions: Structure, Dynamics, and Carbohydrate Affinity of a Lectin Engineered with Fluorinated Tryptophan Analogs.

Protein-carbohydrate interactions play crucial roles in biology. Understanding and modifying these interactions is of major interest for fighting many diseases. We took a synthetic biology approach and incorporated noncanonical amino acids into a bacterial lectin to modulate its interactions with carbohydrates. We focused on tryptophan, which is prevalent in carbohydrate binding sites. The exchange of the tryptophan residues with analogs fluorinated at different positions resulted in three distinctly fluorinated variants of the lectin from Ralstonia solanacearum. We observed differences in stability and affinity toward fucosylated glycans and rationalized them by X-ray and modeling studies. While fluorination decreased the aromaticity of the indole ring and, therefore, the strength of carbohydrate-aromatic interactions, additional weak hydrogen bonds were formed between fluorine and the ligand hydroxyl groups. Our approach opens new possibilities to engineer carbohydrate receptors.

Biocatalytic Friedel-Crafts Acylation and Fries Reaction.

The Friedel-Crafts acylation is commonly used for the synthesis of aryl ketones, and a biocatalytic version, which may benefit from the chemo- and regioselectivity of enzymes, has not yet been introduced. Described here is a bacterial acyltransferase which can catalyze Friedel-Crafts C-acylation of phenolic substrates in buffer without the need of CoA-activated reagents. Conversions reach up to >99 %, and various C- or O-acyl donors, such as DAPG or isopropenyl acetate, are accepted by this enzyme. Furthermore the enzyme enables a Fries rearrangement-like reaction of resorcinol derivatives. These findings open an avenue for the development of alternative and selective C-C bond formation methods.