Structure-guided protein engineering of ammonia lyase for efficient synthesis of sterically bulky unnatural amino acids
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ABSTRACT:
SUBMITTER: Ni Z
PROVIDER: S-EPMC10992142 | biostudies-literature | 2021 Oct
REPOSITORIES: biostudies-literature
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Project description:Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe-Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well-defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe-Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical "handle" by which immunoconjugates and multimers can be engineered.
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Project description:Unnatural proteins are crucial biomacromolecules and have been widely applied in fundamental science, novel biopolymer materials, enzymes, and therapeutics. Cell-free protein synthesis (CFPS) system can serve as a robust platform to synthesize unnatural proteins by highly effective site-specific incorporation of unnatural amino acids (UNAAs), without the limitations of cell membrane permeability and the toxicity of unnatural components. Here, we describe a quick and simple method to synthesize unnatural proteins in CFPS system based on Escherichia coli crude extract, with unnatural orthogonal aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii. The superfolder green fluorescent protein (sfGFP) and p-propargyloxyphenylalanine (pPaF) were used as the model protein and UNAA. The synthesis of unnatural sfGFPs was characterized by microplate spectrophotometer, affinity chromatography, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). This protocol provides a detailed procedure guiding how to use the powerful CFPS system to synthesize unnatural proteins on demand.
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Project description:Current biosynthetic methods for producing proteins containing site-specifically incorporated unnatural amino acids are inefficient because the majority of the amino acid goes unused. Here we present a universal approach to improve the efficiency of such processes using condensed Escherichia coli cultures.
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Project description:Amber suppression has been widely used to incorporate unnatural amino acids (UNAAs) with unique structures or functional side-chain groups into specific sites of the target protein, which expands the scope of protein-coding chemistry. However, this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein, which limits the development of unnatural proteins. To address this challenge, the suppression method using multiple termination codons (TAG, TAA or TGA) was proposed, and cell-free unnatural protein synthesis (CFUPS) system was employed. By the analysis of incorporating 3 different UNAAs (p-propargyloxy-l-phenylalanine, p-azyl-phenylalanine and L-4-Iodophenylalanine) and mass spectrometry, the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation. The CFUPS conditions were further optimized for better UNAA incorporation efficiency, including the orthogonal translation system (OTS) components, magnesium ions, and the redox environment. This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs, thereby synthesizing unnatural proteins with novel physicochemical functions.
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Project description:The introduction of steric bulk to the bidentate ligand in [Ru(tpy)(bpy)(py)](2+) (1; tpy = 2,2':2',6?-terpyridine; bpy = 2,2'-bipyridine; py = pyridine) to provide [Ru(tpy)(Me2bpy)(py)](2+) (2; Me2bpy = 6,6'-dimethyl-2,2'-bipyridine) and [Ru(tpy)(biq)(py)](2+) (3; biq = 2,2'-biquinoline) facilitates photoinduced dissociation of pyridine with visible light. Upon irradiation of 2 and 3 in CH3CN (?irr = 500 nm), ligand exchange occurs to produce the corresponding [Ru(tpy)(NN)(NCCH3)](2+) (NN = Me2bpy, biq) complex with quantum yields, ?500, of 0.16(1) and 0.033(1) for 2 and 3, respectively. These values represent an increase in efficiency of the reaction by 2-3 orders of magnitude as compared to that of 1, ?500 < 0.0001, under similar experimental conditions. The photolysis of 2 and 3 in H2O with low energy light to produce [Ru(tpy)(NN)(OH2)](2+) (NN = Me2bpy, biq) also proceeds rapidly (?irr > 590 nm). Complexes 1-3 are stable in the dark in both CH3CN and H2O under similar experimental conditions. X-ray crystal structures and theoretical calculations highlight significant distortion of the planes of the bidentate ligands in 2 and 3 relative to that of 1. The crystallographic dihedral angles defined by the bidentate ligand, Me2bpy in 2 and biq in 3, and the tpy ligand were determined to be 67.87° and 61.89°, respectively, whereas only a small distortion from the octahedral geometry is observed between bpy and tpy in 1, 83.34°. The steric bulk afforded by Me2bpy and biq also result in major distortions of the pyridine ligand in 2 and 3, respectively, relative to 1, which are believed to weaken its ?-bonding and ?-back-bonding to the metal and play a crucial role in the efficiency of the photoinduced ligand exchange. The ability of 2 and 3 to undergo ligand exchange with ?irr > 590 nm makes them potential candidates to build photochemotherapeutic agents for the delivery of drugs with pyridine binding groups.
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Project description:Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.
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Project description:The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.
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Project description:We describe herein a two-step process for the conversion of serine to a wide array of optically pure unnatural amino acids. This method utilizes a photocatalytic cross-electrophile coupling between a bromoalkyl intermediate and a diverse set of aryl halides to produce artificial analogues of phenylalanine, tryptophan, and histidine. The reaction is tolerant of a broad range of functionalities and can be leveraged toward the scalable synthesis of valuable pharmaceutical scaffolds via flow technology.
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