Site-specific incorporation of PEGylated amino acids into proteins using nonnatural amino acid mutagenesis

Site-directed incorporation of PEGylated nonnatural amino acids with 4, 8, and 12 repeated ethylene glycol units was examined in a cell-free translation system. PEGylated aminophenylalanine derivatives were successfully incorporated into proteins, whereas PEGylated lysines were not. The incorporation efficiency of the PEGylated amino acids decreased with an increase in PEG chain length. The present method will be useful for preparation of proteins which are PEGylated in a site-specific and quantitative manner.     

Site-specific mutagenesis with unnatural amino acids

The incorporation of unnatural amino acids into proteins by site-specific mutagenesis provides a valuable new methodology for the generation of novel proteins that possess unique structural and functional features.     

Site-specific incorporation of unnatural amino acids into urate oxidase in Escherichia coli

Urate oxidase catalyzes the oxidation of uric acid with poor solubility to produce 5-hydroxyisourate and allantoin. Since allantoin is excreted in vivo, urate oxidase has the potential to be a therapeutic target for the treatment of gout. However, its severe immunogenicity limits its clinical application. Furthermore, studies on the structure-function relationships of urate oxidase have proven difficult. We developed a method for genetically incorporating p-azido-L-phenylalanine into target protein in Escherichia coli in a site-specific manner utilizing a tyrosyl suppressor tRNA/aminoacyl-tRNA synthetase system. We substituted p-azido-L-phenylalanine for Phe(170) or Phe(281) in urate oxidase. The products were purified and their enzyme activities were analyzed. In addition, we optimized the system by adding a "Shine-Dalgarno (SD) sequence" and tandem suppressor tRNA. This method has the benefit of site-specifically modifying urate oxidase with homogeneous glycosyl and PEG derivates, which can provide new insights into structure-function relationships and improve pharmacological properties of urate oxidase.     

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.     

Site-specific PEGylation of proteins containing unnatural amino acids

Here, we report a generally applicable PEGylation methodology based on the site-specific incorporation of para-azidophenylalanine into proteins in yeast. The azido group was used in a mild [3+2] cycloaddition reaction with an alkyne derivatized PEG reagent to afford selectively PEGylated protein. This strategy should be useful for the generation of selectively PEGylated proteins for therapeutic applications.     

Efficient incorporation of unnatural amino acids into proteins in Escherichia coli

We have developed a single-plasmid system for the efficient bacterial expression of mutant proteins containing unnatural amino acids at specific sites designated by amber nonsense codons. In this system, multiple copies of a gene encoding an amber suppressor tRNA derived from a Methanocaldococcus jannaschii tyrosyl-tRNA (MjtRNATyrCUA) are expressed under control of the proK promoter and terminator, and a gene encoding the desired mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) is expressed under control of a mutant glnS (glnS') promoter.     

Genetic incorporation of unnatural amino acids into proteins in mammalian cells

We developed a general approach that allows unnatural amino acids with diverse physicochemical and biological properties to be genetically encoded in mammalian cells. A mutant Escherichia coli aminoacyl-tRNA synthetase (aaRS) is first evolved in yeast to selectively aminoacylate its tRNA with the unnatural amino acid of interest. This mutant aaRS together with an amber suppressor tRNA from Bacillus stearothermophilus is then used to site-specifically incorporate the unnatural amino acid into a protein in mammalian cells in response to an amber nonsense codon. We independently incorporated six unnatural amino acids into GFP expressed in CHO cells with efficiencies up to 1 mug protein per 2 x 10(7) cells; mass spectrometry confirmed a high translational fidelity for the unnatural amino acid. This methodology should facilitate the introduction of biological probes into proteins for cellular studies and may ultimately facilitate the synthesis of therapeutic proteins containing unnatural amino acids in mammalian cells.     

Site-specific labeling of proteins with NMR-active unnatural amino acids

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.     

Characteristic amino acid combinations in olfactory G protein-coupled receptors

The human olfactory subgenome has recently been fully characterized with over 1000 genes. Although as many as two thirds of them are expected to be pseudogenes, it still leaves us with about half of all human G protein-coupled receptors being olfactory. It is therefore of great interest to characterize olfactory receptors with high precision. Usually it is done through sequence motifs that are not fully conserved, making an exact characterization difficult. In this paper, we propose a rule-based characterization of olfactory receptors derived from a multiple sequence alignment of human GPCRs. We show that just seven alignment sites are sufficient to characterize 99% of human olfactory GPCRs with one feature, a tyrosine at site 7.41, being of particular importance. We also show dependencies between sites near the extracellular and intracellular region of a membrane-embedded receptor, indicating that olfactory receptors are characterized by a combination of important residues in these two areas, whereas nonolfactory receptors tend to have residues of lower importance at the same sites.     

Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells

In this study we demonstrate that polysialyltransferases are capable of accepting unnatural substrates in terminally differentiated human neurons. Polysialyltransferases catalyze the glycosylation of the neural cell adhesion molecule (NCAM) with polysialic acid (PSA). The unnatural sialic acid analog, N-levulinoyl sialic acid (SiaLev), was incorporated into cell surface glycoconjugates including PSA by the incubation of cultured neurons with the metabolic precursor N-levulinoylmannosamine (ManLev). The ketone group within the levulinoyl side chain of SiaLev was then used as a chemical handle for detection using a biotin probe. The incorporation of SiaLev residues into PSA was demonstrated by protection from sialidases that can cleave natural sialic acids but not those bearing unnatural N-acyl groups. The presence of SiaLev groups on the neuronal cell surface did not impede neurite outgrowth or significantly affect the distribution of PSA on neuronal compartments. Since PSA is important in neural plasticity and development, this mechanism for modulating PSA structure might be useful for functional studies.     

Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo.

The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection.     

Introduction of unnatural amino acids into proteins using expressed protein ligation

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.     

Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids

We have improved the incorporation of l- and d-forms of unnatural amino acid (UAA) N^ε-thiaprolyl-l-lysine (ThzK) into ubiquitin (UB) and green fluorescent protein (GFP) by 2–6 folds with the use of the methylester forms of the UAAs in E coli cell culture. We also improved the yields of UAA-incorporated UB and GFP with the methylester forms of N^ε-Boc-l-Lysine (BocK) and N^ε-propargyl-l-Lysine (PrK) by 2–5 folds compared to their free acid forms. Our work demonstrated that using methylester-capped UAAs for protein expression is a useful strategy to enhance the yields of UAA-incorporated proteins.     

游离脂肪酸受体蛋白研究进展

游离脂肪酸不仅是人和动物体的一种重要能量来源,也是一种重要的信号分子。最近研究表明,游离脂肪酸受体蛋白在维持机体内的葡萄糖稳衡、脂肪形成、白细胞功能等生理过程中都有重要的作用,对于调控人或动物的营养代谢及疾病发生具有重要生理意义。     

G protein-coupled receptors for free fatty acids

Free fatty acids (FFAs) are not only an important direct source of energy but they also play key roles in regulating a range of physiological responses. Although it was long assumed that such effects of FFAs must occur following cellular uptake, and potentially via their conversion to fatty acyl-CoAs, it is now apparent that FFAs also function directly as agonists at a number of G protein-coupled receptors (GPCRs). Tissue distribution studies and, subsequently, siRNA knock-down experiments have indicated key roles for these GPCRs in glucose homeostasis, adipogenesis, white cell recruitment and potentially in a range of other processes. Considerable interest is thus now centred on the generation of potent and selective small molecule ligands, both as tool compounds to further unravel the biology and physiological role of this group of GPCRs and as starting points for possible therapeutic intervention in a range of areas, particularly those associated with 'metabolic syndrome'.     

Site-specific solvent exposure analysis of a membrane protein using unnatural amino acids and F nuclear magnetic resonance

Membrane proteins play an essential role in cellular metabolism, transportation and signal transduction across cell membranes. The scarcity of membrane protein structures has thus far prevented a full understanding of their molecular mechanisms. Preliminary topology studies and residue solvent exposure analysis have the potential to provide valuable information on membrane proteins of unknown structure. Here, a ¹⁹F-containing unnatural amino acid (trimethylfluoro-phenylalanine, tfmF) was applied to accomplish site-specific ¹⁹F spin incorporation at different sites in diacylglycerol kinase (DAGK, an Escherichia coli membrane protein) for site-specific solvent exposure analysis. Due to isotope effect on ¹⁹F spins, a standard curve for ¹⁹F-tfmF chemical shifts was drawn for varying solvent H₂O/D₂O ratios. Further site-specific ¹⁹F solvent isotope shift analysis was conducted for DAGK to distinguish residues in water-soluble loops, interfacial areas or hydrophobic membrane regions. This site-specific solvent exposure analysis method could be applied for further topological analysis of other membrane proteins.     

Introduction of unnatural amino acids into chalcone isomerase.

The active site cysteine residue of chalcone isomerase was rapidly and selectively modified under denaturing conditions with a variety of electrophilic reagents. These denatured and modified enzyme were renatured to produce enzyme derivatives containing a series of unnatural amino acids in the active site. Addition of methyl, ethyl, butyl, heptyl, and benzyl groups to the cysteine sulfur does not abolish catalytic activity, although the activity decreases as the steric bulk of the amino acid side-chain increases. Modification of the cysteine to introduce a charged homoglutamate or a neutral homoglutamine analogue results in retention of 22% of the catalytic activity. Addition of a methylthio group (SMe) to the cysteine residue of native chalcone isomerase preserves 85% of the catalytic activity measured with 2',4',4-trihydroxychalcone, 2',4',6',4-tetrahydroxychalcone, or 2'-hydroxy-4-methoxychalcone as substrates. The competitive inhibition constant for 4',4-dihydroxychalcone, the substrate inhibition constant for 2',4',4-trihydroxychalcone, and other steady-state kinetic parameters for the methanethiolated enzyme are very similar to those of the native enzyme. The strong binding of 4',4-dihydroxychalcone to the methanethiolated enzyme shows that there is no steric repulsion between this modified amino acid residue and the substrate analogue. This structure-activity study clearly demonstrates that the active site cysteine residue does not function as an acid-base or nucleophilic group in producing the catalysis or substrate inhibition observed with chalcone isomerase. The method presented in this paper allows for the rapid introduction of a series of unnatural amino acids into the active site as a means of probing the structure-function relationship.     

Site-specific incorporation of non-natural amino acids into proteins in mammalian cells with an expanded genetic code

We describe a detailed protocol for incorporating non-natural amino acids, 3-iodo-L-tyrosine (IY) and p-benzoyl-L-phenylalanine (pBpa), into proteins in response to the amber codon (the UAG stop codon) in mammalian cells. These amino acids, IY and pBpa, are applicable for structure determination and the analysis of a network of protein-protein interactions, respectively. This method involves (i) the mutagenesis of the gene encoding the protein of interest to create an amber codon at the desired site, (ii) the expression in mammalian cells of the bacterial pair of an amber suppressor tRNA and an aminoacyl-tRNA synthetase specific to IY or pBpa and (iii) the supplementation of the growth medium with these amino acids. The amber mutant gene, together with these bacterial tRNA and synthetase genes, is introduced into mammalian cells. Culturing these cells for 16-40 h allows the expression of the full-length product from the mutant gene, which contains the non-natural amino acid at the introduced amber position. This method is implemented using the conventional tools for molecular biology and treating cultured mammalian cells. This protocol takes 5-6 d for plasmid construction and 3-4 d for incorporating the non-natural amino acids into proteins.