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Human serum albumin (HSA) has been used to extend the serum half-life of therapeutic proteins owing to its exceptionally long serum half-life via the neonatal Fc receptor (FcRn)-mediated recycling mechanism. In most cases, only one HSA molecule was conjugated to a therapeutic protein, leading to a limited extension of the serum half-life. In this study, we hypothesized that conjugation of multiple HSA molecules to a therapeutic protein significantly further extends the serum half-life via multivalent HSA-FcRn interactions. We chose urate oxidase (Uox), a tetrameric therapeutic protein used for the treatment of gout, as a model. In previous studies, only one HSA molecule was site-specifically conjugated to one Uox because of poor conjugation yield of the relatively slow bio-orthogonal chemistry, strain-promoted azide-alkyne cycloaddition (SPAAC). To increase the number of HSA molecules conjugated to one Uox, we employed the faster bio-orthogonal chemistry, inverse electron demand Diels-Alder reaction (IEDDA). We site-specifically introduced the phenylalanine analog with a fast-reacting tetrazine group (frTet) into position 174 of each subunit of Uox. We then achieved site-specific HSA conjugation to each subunit of Uox via IEDDA, generating Uox conjugated to four HSA molecules (Uox-HSA4), with a small portion of Uox conjugated to three HSA molecules (Uox-HSA3). We characterized Uox-HSA4 as well as Uox variants conjugated to one or two HSA molecules prepared via SPAAC (Uox-HSA1 or Uox-HSA2). The enzyme activity of all three Uox-HSA conjugates was comparable to that of unmodified Uox. We found out that an increase in HSA molecules conjugated to Uox (multiple albumin-conjugated therapeutic protein) enhanced FcRn binding and consequently prolonged the serum half-life in vivo. In particular, the conjugation of four HSA molecules to Uox led to a prominent extension of the serum half-life (over 21 h), which is about 16-fold longer than that of Uox-WT.

Oxidoreductases have been used for the synthesis of value-added chemicals via redox reactions under mild conditions. However, these enzymes often require cofactors, which are expensive and small mo...

An inverse-electron-demand Diels-Alder (IEDDA) reaction using genetically encoded tetrazine variants enables rapid bioconjugation for diverse applications in vitro and in cellulo. However, in vivo bioconjugation using genetically encoded tetrazine variants is challenging, because the IEDDA coupling reaction competes with rapid elimination of reaction partners in vivo. Here, we tested the hypothesis that a genetically encoded phenylalanine analogue containing a hydrogen-substituted tetrazine (frTet) would increase the IEDDA reaction rate, thereby allowing for successful bioconjugation in vivo. We found that the in vitro IEDDA reaction rate of superfolder green fluorescent protein (sfGFP) containing frTet (sfGFP-frTet) was 12-fold greater than that of sfGFP containing methyl-substituted tetrazine (sfGFP-Tet_v2.0). Additionally, sfGFP variants encapsulated with chitosan-modified, pluronic-based nanocarriers were delivered into nude mice or tumor-bearing mice for in vivo imaging. The in vivo-delivered sfGFP-frTet exhibited almost complete fluorescence recovery upon addition of trans-cyclooctene via the IEDDA reaction within 2 h, whereas sfGFP-Tet_v2.0 did not show substantial fluorescence recovery. These results demonstrated that the genetically encoded frTet allows an almost complete IEDDA reaction in vivo upon addition of trans-cyclooctene, enabling temporal control of in vivo bioconjugation in a very high yield.

Enzyme immobilization is very important for diverse enzyme applications. Particularly, there is a growing need for coimmobilization of multiple enzymes for biosensing and synthetic applications. However, it is still challenging to coimmobilize two enzymes with desirable features, including high immobilization yield, retention of enzymatic activity, and low leaching. In this study, we demonstrated that a pluronic-based nanocarrier (PNC) can be an encapsulation platform for immobilization of various single enzymes. Since the PNC is temperature-sensitive, a simple temperature change from 4 to 37 °C led to a substantial size reduction and enzyme encapsulation. All six enzymes tested were encapsulated by the PNC in high yield (∼90%) with the retained enzymatic activity (95%). The leaching of encapsulated enzymes was very minimal (0.13% for 2 weeks). Then, we demonstrated that the PNC can efficiently coencapsulate two enzymes, formate dehydrogenase (FDH) and mannitol dehydrogenase (MDH), for a cascade reaction producing d-mannitol. Coencapsulation of FDH and MDH resulted in an over 10-fold increase in d-mannitol production compared to the free mix of FDH and MDH, likely due to the enhanced local concentrations of FDH and MDH inside the PNC.

Therapeutic proteins are indispensable for treatment of various human diseases. However, intrinsic short serum half-lives of proteins are still big hurdles for developing new therapeutic proteins or expanding applications of existing ones. Urate oxidase (Uox) is a therapeutic protein clinically used for treatment of hyperuricemia. Due to its short half-life, its application for gout treatment requires prolonging the half-life in vivo. Conjugation of a fatty acid (FA), a serum albumin (SA) ligand, to therapeutic proteins/peptides is an emerging strategy to prolong serum half-life presumably via neonatal Fc receptor (FcRn)–mediated recycling. FA conjugation was proven effective for peptides and small proteins (less than 28 kDa), but not for Uox (140 kDa). We hypothesized that the intramolecular distance in the conjugate of FA and Uox is a critical factor for effective FcRn-mediated recycling. In order to control the intramolecular distance in the conjugate, we varied linker lengths between Uox and palmitic acid (PA). There was a linear correlation between the linker length and serum half-life of PA–conjugated Uox (Uox-PA) conjugates. The longer linker led to about 7-fold greater extension of serum half-life of Uox in mice than the unmodified Uox. The trend in serum half-life extension matched well with that in the tertiary structure formation of FcRn/SA/Uox-PA in vitro. These results demonstrate that the intramolecular distance in the conjugate of Uox and FA governs the stable formation of FcRn/SA/FA-conjugated protein and serum half-life extension in vivo. These findings would also contribute to development of effective FAconjugated therapeutic proteins.

Glucagon-like peptide-1 (GLP-1) is a peptide hormone with tremendous therapeutic potential for treating type 2 diabetes mellitus. However, the short half-life of its native form is a significant drawback. We previously prolonged the plasma half-life of GLP-1 via site-specific conjugation of human serum albumin (HSA) at position 16 of recombinant GLP-1 using site-specific incorporation of p-azido-phenylalanine (AzF) and strain-promoted azide-alkyne cycloaddition (SPAAC). However, the resulting conjugate GLP1_8G16AzF-HSA showed only moderate in vivo glucose-lowering activity, probably due to perturbed interactions with GLP-1 receptor (GLP-1R) caused by the albumin-linker. To identify albumin-conjugated GLP-1 variants with enhanced in vivo glucose-lowering activity, we investigated the conjugation of HSA to a C-terminal region of GLP-1 to reduce steric hindrance by the albumin-linker using two different conjugation chemistries. GLP-1 variants GLP1_8G37AzF-HSA and GLP1_8G37C-HSA were prepared using SPAAC and Michael addition, respectively. GLP1_8G37C-HSA exhibited a higher glucose-lowering activity in vivo than GLP1_8G16AzF-HSA, while GLP1_8G37AzF-HSA did not. Another GLP-1 variant, GLP1_8A37C-HSA, had a glycine to alanine mutation at position 8 and albumin at its C-terminus and exhibited in vivo glucose-lowering activity comparable to that of GLP1_8G37C-HSA, despite a moderately shorter plasma half-life. These results showed that site-specific HSA conjugation to the C-terminus of GLP-1 via Michael addition could be used to generate GLP-1 variants with enhanced glucose-lowering activity and prolonged plasma half-life in vivo.

The number of therapeutic peptides for human treatment is growing rapidly. However, their development faces two major issues: the poor yield of large peptides from conventional solid-phase synthesis, and the intrinsically short serum half-life of peptides. To address these issues, we investigated a platform for the production of a recombinant therapeutic peptide with an extended serum half-life involving the site-specific conjugation of human serum albumin (HSA). HSA has an exceptionally long serum half-life and can be used to extend the serum half-lives of therapeutic proteins and peptides. We used glucagon-like-peptide 1 (GLP-1) as a model peptide in the present study. A &ldquo, clickable&rdquo, non-natural amino acid&mdash, p-azido-l-phenylalanine (AzF)&mdash, was incorporated into three specific sites (V16, Y19, and F28) of a GLP-1 variant, followed by conjugation with HSA through strain-promoted azide&ndash, alkyne cycloaddition. All three HSA-conjugated GLP-1 variants (GLP1_16HSA, GLP1_19HSA, and GLP1_28HSA) exhibited comparable serum half-lives in vivo. However, the three GLP1_HSA variants had different in vitro biological activities and in vivo glucose-lowering effects, demonstrating the importance of site-specific HSA conjugation. The platform described herein could be used to develop other therapeutic peptides with extended serum half-lives.

Ophthalmic formulations have classically been administered to eyes through eye drops, which have poor delivery efficiency and require frequent instillation. As a means of overcoming these drawbacks, ocular-drug delivery via therapeutic contact lenses has caused great interest. A simple way to prepare therapeutic contact lenses is to immerse lenses in concentrated drug solution. However, not all ocular drugs can be efficiently loaded into contact lenses by a simple soaking. In particular, our previous study showed that two antibacterial ocular drugs, ofloxacin (OFX) and neomycin (NEO), were poorly loaded to poly(2-hydroxyethyl methacrylate) (pHEMA)-based contact lenses. Herein, we investigated whether alteration of lens composition using several negatively charged comonomers can enhance loading of ocular drugs with poor loadability (OFX and NEO). As comonomers, methacrylic acid (MAA), acrylic acid (AA), and 4-methyl-4-pentenoic acid (MPA) were used, generating p(HEMA-co-MAA), p(HEMA-co-AA), and p(HEMA-co-MPA) hydrogel-based contact lenses, respectively. Contact lenses containing comonomers exhibited an increase in loading of OFX and NEO. In particular, compared with pHEMA contact lenses, contact lenses containing 2.5 mol% AA exhibited enhanced loading of OFX and NEO, 18 and 53 times, respectively. Charge interactions between comonomers and the drug were considered primary factors in the substantial increase in drug loading.

Soft contact lenses made with poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels modified with various comonomers such as N-vinyl pyrrolidone, methacrylic acid, glycidyl methacrylate, and glycerol monomethacrylate were prepared to investigate the effect of adding the comonomer on the water content, surface wettability, and tensile modulus. These polymers were synthesized by the free radical polymerization of 2-hydroxyethyl methacrylate (HEMA) with the comonomers in the presence of divinyl benzene used as the crosslinker and azobisisobutyronitrile as the initiator. The chemical structure and transmittance of the hydrogels were analyzed by FTIR and UV/Vis spectrophotometers. The surface wettability and tensile modulus were also studied by measuring the contact angle and tensile modulus with a universal testing machine (UTM). Regarding the properties of water in the hydrogels, the ratio between free to bound water was investigated using differential scanning calorimetry (DSC). As the concentration of crosslinker in the hydrogels increases, the tensile strength also increases, whereas the internal water content and contact angle decrease. The effect of the comonomer composition of the hydrogels was also investigated to optimize the various properties of these comonomers for soft contact lenses.

Enzymes are biocatalysts that play key roles in diverse chemical reactions in living organisms and industrial conversion processes generating value-added products. Since wild-type enzymes obtained from nature are normally not optimal for various applications, enzyme engineering is usually required for enhanced or new properties. Site-specific incorporation of a non-natural amino acid became a powerful protein-engineering tool. In this short review, we briefly summarize our contribution to enzyme complex formation and active site design of enzymes using the technique of site-specific incorporation of a non-natural amino acid. First, site-specific incorporation of a non-natural amino acid at a permissive site of an enzyme led to bioconjugation to other molecules without compromising critical properties. Murine dihydrofolate reductase (mDHFR) was site-specifically conjugated to a biotin via click chemistry to achieve site-specific immobilization. Similarly, formate dehydrogenase (FDH) was site-specifically c...

Mussel adhesive proteins (MAPs) have great potential as bioglues, particularly in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows for production of Dopa-incorporated bioengineered MAPs (dMAPs), the low production yield hinders the practical application of dMAPs. This low production yield of dMAPs is due to low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, to enhance the production yield of dMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). To use the Dopa-specific Methanococcus jannaschii TyrRS (MjTyrRS-Dopa), we altered the anticodon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyr AUA ) to recognize a tyrosine codon (AUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyr AUA increased the production yield of Dopa-incorporated MAP foot protein type 3 (dfp-3) by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of dfp-3. Even with coexpression of Dopa-recognizing TyrRSs, dfp-3 has a high Dopa incorporation yield (over 90%) compared to ones prepared without TyrRS coexpression.

Hypoxia, resulting from the imbalance between oxygen supply and consumption is a critical component of the tumor microenvironment. It has a paramount impact on cancer growth, metastasis and has long been known as a major obstacle for cancer therapy. However, none of the clinically approved anticancer therapeutics currently available for human use directly tackles this problem. Previous clinical trials of targeting tumor hypoxia with bioreductive prodrugs have failed to demonstrate satisfactory results. Therefore, new ideas are needed to overcome the hypoxia barrier. The method of modulating hypoxia to improve the therapeutic activity is of great interest but remains a considerable challenge. One of the emerging concepts is to supply or generate oxygen at the tumor site to increase the partial oxygen pressure and thereby reverse the hypoxia and its effects. In this review, we present an overview of the recent progress in the development of novel nanomaterials for the alleviation of hypoxic microenvironment. Two main strategies for hypoxia augmentation, i) direct delivery of O2 into the tumor, and ii) in situ O2 generations in the tumor microenvironment through different methods such as catalytic decomposition of endogenous hydrogen peroxide (H2O2) and light-triggered water splitting are discussed in detail. At present, these emerging nanomaterials are in their early phase and expected to grow rapidly in the coming years. Despite the promising start, there are several challenges needed to overcome for successful clinical translation.

Therapeutic proteins are indispensable in the treatment of various human diseases. Despite the many benefits of therapeutic proteins, they also exhibit diverse side effects. Therefore, reducing unwanted side effects of therapeutic proteins as well as enhancing their therapeutic efficacy are very important in developing therapeutic proteins. Urate oxidase (UOX) is a therapeutic enzyme that catalyzes the conversion of uric acid (UA) into a soluble metabolite, and it is used clinically for the treatment of hyperuricemia. Since UA degradation by UOX generates H2O2 (a cytotoxic side product), UOX was co-delivered with catalase-mimic nanoparticles (AuNPs) using biocompatible pluronic-based nanocarriers (NCs) to effectively reduce H2O2-associated toxicity in cultured cells and to enhance UA degradation efficiency in vivo. Simple temperature-dependent size changes of NCs allowed co-encapsulation of both UOX and AuNPs at a high loading efficiency without compromising critical properties, resulting in efficient modulation of a mixing ratio of UOX and AuNPs encapsulated in NCs. Co-localizing UOX and AuNPs in the NCs led to enhanced UA degradation and H2O2 removal in vitro, leading to a great reduction in H2O2-associated cytotoxicity compared with UOX alone or a free mixture of UOX and AuNPs. Furthermore, we demonstrated that co-delivery of UOX and AuNPs using NCs significantly improves in vivo UA degradation compared to simple co-injection of free UOX and AuNPs. More broadly, we showed that biocompatible pluronic-based nanocarriers can be used to deliver a target therapeutic protein along with its toxicity-eliminating agent in order to reduce side effects and enhance efficacy.

An internally duplicated α-type carbonic anhydrase (CA) from Dunaliella species (Dsp-CA) has been expressed as single-domain derivatives, N- and C- half domains. Although both derivatives have structures similar to that of a known CA (PDB ID: 1y7w ), only the C-half domain (Dsp-CA-c) exhibited enzymatic activity, albeit with low solubility. In order to increase the solubility of Dsp-CA-c, its proximal N-terminal amino acids 1–11 were substituted with VSEPHDYNYEK (NT11) of the N-half domain (Dsp-CA-n) which exhibits a high soluble expression yield. The new chimeric gene products, Dsp-nCA-c displayed approximately 2-fold the solubility and activity shown by Dsp-CA-c. Dsp-nCA-c showed increased thermal stability (ΔTm > 10 °C) by multimerization compared to Dsp-CA-c. Finally, the chimeric protein effectively catalyzed the conversion of CO2 into its calcite form in the presence of CaCl2. These findings indicate that the substituted NT11 may contribute to soluble expression and enhanced activity of Dsp-nCA-c and cause multimerization which can confer increased thermostability to Dsp-nCA-c. Therefore, N-terminal engineering can be an effective strategy for improving soluble production yield and thermostabillity of CA without disrupting catalytic activity, and the engineered CA could be usefully employed for the development of an efficient enzymatic CO2 sequestration system.

The ability to incorporate non-natural amino acids during protein biosynthesis has greatly broadened the amino acid sequence space available for protein engineering. The increasing number of non-natural amino acids provides novel sidechain chemistries, structures, and functionalities for engineered proteins including enzymes. Therefore, understanding how non-natural amino acid incorporation (non-natural amino acid mutagenesis) affects protein function is crucial. This study investigates the positional effects of hydrophobic non-natural amino acid mutagenesis of the enzyme surface on the enzyme structure and function with the ultimate goal of identification of permissive sites for non-natural amino acid mutagenesis. A bulky, hydrophobic non-natural amino acid, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at the solvent-exposed surface of a murine dihydrofolate reductase (mDHFR) enzyme. Incorporation of 2Nal was performed at six solvent-exposed sites (V43, E44, F142, E143, F179, and E180), generating six mDHFR variants. Incorporation of 2Nal into F142 or F179 did not exhibit any substantial change in the secondary structure and catalytic activities, whereas 2Nal incorporation at hydrophilic Glu sites significantly changed the secondary structure and catalytic activities. Such different responses upon 2Nal incorporation at hydrophobic and hydrophilic residues were likely due to the structural changes caused by the hydrophobicity change upon mutation. A non-conservative mutation of V43 with 2Nal significantly reduced the catalytic activities suggesting that a substantial size change of sidechain also causes the structural changes. These results suggest that even hydrophobic, bulky non-natural amino acids can be incorporated at the enzyme surface without compromising the enzymatic activities.

Polyethylene glycol (PEG) has been widely used as a serum half-life extender of therapeutic proteins. However, due to immune responses and low degradability of PEG, developing serum half-life extender alternatives to PEG is required. Human serum albumin (HSA) has several beneficial features as a serum half-life extender, including a very long serum half-life, good degradability, and low immune responses. In order to further evaluate the efficacy of HSA, we compared the extent of serum half-life extension of a target protein, superfolder green fluorescent protein (sfGFP), upon HSA conjugation with PEG conjugation side-by-side. Combination of site-specific incorporation of p-azido-l-phenylalanine into sfGFP and copper-free click chemistry achieved the site-specific conjugation of a single HSA, 20 kDa PEG, or 30 kDa PEG to sfGFP. These sfGFP conjugates exhibited the fluorescence comparable to or even greater than that of wild-type sfGFP (sfGFP-WT). In mice, HSA-conjugation to sfGFP extended the serum half-life 9.0 times compared to that of unmodified sfGFP, which is comparable to those of PEG-conjugated sfGFPs (7.3 times for 20 kDa PEG and 9.5 times for 30 kDa PEG). These results clearly demonstrated that HSA was as effective as PEG in extending the serum half-life of a target protein. Therefore, with the additional favorable features, HSA is a good serum half-life extender of a (therapeutic) protein as an alternative to PEG.

Reducing amyloid-β (Aβ) accumulation is a promising strategy for developing Alzheimer’s Disease (AD) therapeutics. We recently reported that a triphenylmethane food dye analog, Brilliant Blue G (BBG), is a dose-dependent modulator of in vitro amyloid-β aggregation and cytotoxicity in cell-based assays. Following up on this recent work, we sought to further evaluate this novel modulator in a therapeutically-relevant AD transgenic mouse model. BBG was orally administered to APPSwDI/NOS2-/- mice for three months in order to assess its biocompatibility, its permeability across the blood-brain barrier, and its efficacy at rescuing AD pathology. The results showed that BBG was well-tolerated, caused no significant weight change/unusual behavior, and was able to significantly cross the AD blood-brain barrier in APPSwDI/NOS2-/- mice. Immunohistochemical and electron microscopic analysis of the brain sections revealed that BBG was able to significantly prevent neuronal loss and reduce intracellular APP/Aβ in hippocampal neurons. This is the first report of 1) the effect of Brilliant Blue G on neuronal loss in a transgenic animal model of AD, 2) oral administration of BBG to affect a protein conformation/aggregation disease, and 3) electron microscopic ultrastructural analysis of AD pathology in APPSwDI/NOS2-/- mice.

Conjugation of serum albumin or one of its ligands (such as fatty acid) has been an effective strategy to prolong the serum half-lives of drugs via neonatal Fc receptor (FcRn)&ndash, mediated recycling of albumin. So far, fatty acid (FA) has been effective in prolonging the serum half-lives for therapeutic peptides and small proteins, but not for large therapeutic proteins. Very recently, it was reported a large protein conjugated to FA competes with the binding of FcRn with serum albumin, leading to limited serum half-life extension, because primary FA binding sites in serum albumin partially overlap with FcRn binding sites. In order to prevent such competition, longer linkers between FA and the large proteins were required. Herein, we hypothesized that small proteins do not cause substantial competition for FcRn binding to albumin, resulting in the extended serum half-life. Using a small protein (28 kDa), we investigated whether the intramolecular distance in FA-protein conjugate affects the FcRn binding with albumin and serum half-life using linkers with varying lengths. Unlike with the FA-conjugated large protein, all FA-conjugated small proteins with different linkers exhibited comparable the FcRn binding to albumin and extended serum half-life.

Human serum albumin (HSA) has been investigated as a serum half-life extender of therapeutic proteins thanks to its unusually long serum half-life. However, in mice, the serum half-life of a HSA-conjugated protein was much shorter than that of HSA in humans, likely due to the species-dependent nature of albumin–FcRn interactions. Herein, we investigated species-dependent albumin–FcRn interactions using species-matched albumin (mouse serum albumin) and species-mismatched albumin (HSA) in non-transgenic mice. We site-specifically introduced a clickable non-natural amino acid to a target protein followed by conjugation to an albumin species via a hetero-bifunctional linker. Using in vitro binding assays, we showed that both HSA- and MSA-conjugated proteins bound mouse FcRns. Conjugation of HSA led to very limited extension of the serum half-life of sfGFP in mice (16.3 h), compared to that of HSA in transgenic mice harboring an allele of mouse FcRn knock-out and expressing humn FcRn (67 h) reported previously. These results suggest that the FcRn-mediated recycling of HSA is not effective in mice. However, conjugation of mouse serum albumin (MSA) resulted in a serum half-life of sfGFP (27.7 h) comparable to that of MSA in mice (28.8 h). Altogether, our study supported that albumin–FcRn interactions are species dependent in vivo.

Polymer conjugation to proteins has been widely used to improve or expand protein properties. However, polymer conjugation to random sites of a target protein often led to a significant loss of critical protein properties. In order to overcome this, polymer conjugation to specific sites of a protein was developed using the site-specific introduction of non-natural amino acids and bioorthogonal chemistries. This review summarizes the recent advances in bioorthogonal chemistries. As the repertoire of bioorthogonal chemistries available for polymer conjugation is expanding, the site-specific polymer conjugation technique would be a valuable platform technique to design novel protein–polymer conjugates.

As the number of reactions requiring biotransformation continues to grow, manipulating the enzyme substrate specificity becomes very important. Complementary to conventional enzyme engineering techniques based on only natural amino acids, we hypothesized that the site-specific incorporation of a non-natural amino acid in vivo into an enzyme can be used to re-design the active site for the altered substrate specificity. To test our hypothesis, we introduced the non-natural amino acid L -2-naphthylalanine (2Nal) into the active site of the model enzyme murine dihydrofolate reductase (mDHFR). We explored whether the substrate specificity of the enzyme could be switched from the good substrate dihydrofolate (DHF) to the poor substrate folate (FOL). We used two protein design programs (RosettaLigand and RosettaDesign) to calculate ligand docking and conformational stability, respectively, for the evaluation of multiples sites in the mDHFR. From the calculations, position 31 was predicted as an optimal 2Nal incorporation site. One mDHFR variant containing 2Nal at position 31 (mDHFR 2Nal31 ) was expressed in the Escherichia coli expression host cells equipped with the engineered yeast phenylalanyl-tRNA and phenylalanyl-tRNA synthetase pair. As expected, the kinetic assays of purified mDHFR variant revealed that mDHFR 2Nal31 has the enhanced binding affinity toward FOL and also exhibits 7.6-fold enhanced catalytic efficiency of FOL over DHF compared to mDHFR WT .

The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering.

Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL × h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons and skeletal muscle atrophy. The majority of ALS cases are acquired spontaneously, with inherited disease accounting for only 10 % of all cases. Recent studies provide compelling evidence that aggregates of misfolded proteins underlie both types of ALS. Small molecules such as artificial chaperones can prevent or even reverse the aggregation of proteins associated with various human diseases. However, their very high active concentration (micromolar range) severely limits their utility as drugs. We synthesized several ester and amide derivatives of chemical chaperones. The lead compound 14, 3-((5-((4,6-dimethylpyridin-2-yl)methoxy)-5-oxopentanoyl)oxy)-N,N-dimethylpropan-1-amine oxide shows, in the micromolar concentration range, both neuronal and astrocyte protective effects in vitro; at daily doses of 10 mg kg(-1) 14 improved the neurological functions and delayed body weight loss in ALS mice. Members of this new chemical chaperone derivative class are strong candidates for the development of new drugs for ALS patients.