Site-specific PEGylation of native disulfide bonds in therapeutic proteins

Native disulfide bonds in therapeutic proteins are crucial for tertiary structure and biological activity and are therefore considered unsuitable for chemical modification. We show that native disulfides in human interferon alpha-2b and in a fragment of an antibody to CD4(+) can be modified by site-specific bisalkylation of the two cysteine sulfur atoms to form a three-carbon PEGylated bridge. The yield of PEGylated protein is high, and tertiary structure and biological activity are retained.     

Posttranslational modification of therapeutic proteins in plants

Plants have emerged as an alternative to current systems for the production of therapeutic proteins. The advantages of plants for the low-cost and large-scale production of safe and biologically active mammalian proteins have been documented recently. A major advantage of transgenic plants over production systems that are based on yeast or Escherichia coli is their ability to perform most of the posttranslational modifications (PTMs) that are required for the bioactivity and pharmacokinetics of recombinant therapeutic proteins. Furthermore, recent advances in the control of PTMs in transgenic plants have made it possible for plants to perform, at least to some extent, human-like modifications of recombinant proteins. Hence, plants have become a suitable alternative to animal cell factories for the production of therapeutic proteins.     

Site-specific lipophilic modification of interferon-alpha

Interferon- (IFN),⁴ a cytokine with modulatory activities on many cell types, is useful for treating many types of cancer and infectious diseases. This study investigates whether modification of a protein, using IFN as an example, with a lipophilic group can alter its distribution and kinetic properties in the body. Ser163 of IFN2a was mutated to Cys to generate a free sulfhydryl group for site-specific chemical modification. IFN2a(S163C) was conjugated by iodoacetamide derivatives of varying lengths, and the modified IFN2a was purified by gel filtration chromatography. The biological activities of IFN2a(S163C) and lipophilized IFN2a(S163C) were similar to that of IFN2a, as evidenced by their inhibitory effects on the growth of Daudi cells and on the replication of vesicular stomatitis virus in Madin-Darby bovine kidney cells. Lipophilized IFN2a(S163C) bound to human serum albumin and cell membranes more readily than did IFN2a. Future experiments will investigate whether lipophilized IFN2a(S163C) has improved pharmacokinetic properties.     

Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification

An often limiting factor for studying protein folding by single-molecule fluorescence resonance energy transfer (FRET) is the ability to site-specifically introduce a photostable organic FRET donor (D) and a complementary acceptor (A) into a polypeptide chain. Using alternating-laser excitation and chymotrypsin inhibitor 2 as a model, we show that chemical labeling of a unique cysteine, followed by enzymatic modification of a reactive glutamine in an N-terminally appended substrate sequence recognition tag for transglutaminase (TGase) affords stoichiometrically D-/A-labeled protein suitable for single-molecule FRET experiments. Thermodynamic data indicate that neither the presence of the TGase tag nor D/A labeling perturbs protein stability. As the N terminus in proteins is typically solvent accessible, a TGase tag can (in principle) be appended to any protein of interest by genetic engineering. Two-step chemical/enzymatic labeling may thus represent a simple, low-cost, and widely available strategy for D/A labeling of proteins for FRET-based single-molecule protein folding studies, even for non-protein-experts laboratories.     

Site-specific protein modification: advances and applications

Although chemical methods to modify proteins in a sequence-specific manner have yet to be developed, site-specific post-translational modification of proteins has recently emerged as a major focus in biological chemistry. Post-translational modification with functionalized substrate analogues opens up several unique avenues to induce selective reactivity into proteins in a sequence-specific manner, and can be applied to protein identification and manipulation in both in vitro and in vivo contexts. Further in vivo applications of this method will enable the imaging of cellular processes, avoiding nonspecific labeling and probe scattering, major complications observed in nonenzymatic methods. Additionally, new tools for in vitro protein modification have been developed that offer more versatile ways to study protein structure and function.     

Site-specific laser modification (cleavage) of oligodeoxynucleotides

Sequence-specific photomodification of oligodeoxynucleotide pAGAGTATTGACTTA ("a target") has been carried out with the aid of complementary fluorescent probes. Such a probe consisted of oligodeoxynucleotide pAATACTCT and a chromophore group attached to its 5' end. Three different derivatives of ethidium bromide were used as a chromophore. The photomodification was induced by nitrogen laser radiation (337 nm, 15 MW/cm2). The irradiation induces the following photodamages: target cleavage at the specific binding site with a cutting off of the 8-mer from its 5' end (yield up to 12%), formation of specific covalent adduct target-probe with a yield of 20-70%, and piperidine-sensitive target modifications with a 7-27% yield (for different chromophores). The total yield of specific photodamages of all kinds is 50-80%. The target cleavage and generation of piperidine-sensitive modifications are optically nonlinear processes. Piperidine treatment of the irradiated samples led to specific cleavage of the target with the yield up to 40%. All kinds of observed modifications are not influenced by high concentrations of free radical scavengers: 1.3M tBuOH and 10 mM cystamine. The pattern of cleavage indicates that the most probable position of the chromophore is between T8 and G9 of the target, i.e., the chromophore stacks on top of the last A.T base pair of the duplex. The aggregate of evidence is in agreement with the mechanism of nonlinear photomodification (the cleavage and generation of piperidine-sensitive modifications) based on the transfer of two-photon excitation energy from the chromophore to the target.     

Site-specific modification of hemoglobin by methyl acetyl phosphate

Methyl acetyl phosphate, which was originally synthesized as a site-specific reagent for hydroxybutyrate dehydrogenase [R. Kluger and W.-C. Tsui (1980) J. Org. Chem. 45, 2723], also has an affinity for the binding site for 2,3-diphosphoglycerate in hemoglobin. Three residues in or near this cleft between the beta-chains are acetylated by this reagent, i.e., Val-1, Lys-82, and Lys-144. There is no detectable acetylation of any of the amino groups of the alpha-chain. These results indicate the specificity of methyl acetyl phosphate in its reaction with hemoglobin.     

Site-specific modification of the lactose operator with acetylaminofluorene

We have synthesized the tetradecamer GAGCXGATAACAAG containing a part of the sequence of the lactose operator. A guanine base in the sequence is replaced by the adduct of the carcinogen 2-acetylaminofluorene with guanine. Under the standard conditions of de-protection, the fluorene moiety is lost, leaving behind a guanine oxidation product. New conditions of de-protection have been developed which allow the isolation of an oligonucleotide containing the adduct of 2-aminofluorene with guanine. The presence of the aminofluorene adduct greatly increases retention on reverse phase chromatography and produces a unique pattern of sequencing bands.     

Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli

The site-specific modification of proteins is expected to be an important capability for the synthesis of bioconjugates in the future. However, the traditional repertoire of reactions available for the direct modification of proteins suffers from lack of specificity, necessitating costly downstream processing to isolate the specific species of interest. (1) Here, we use a well-established, glycan-specific chemistry to PEGylate model glycoproteins, each containing a unique reactive GalNAc attached to a specifically engineered threonine residue. By engineering E. coli to execute the initial steps of human, mucin-type O-glycosylation, we were able to obtain homogeneous site-specifically modified glycoproteins with fully human glycan linkages. Two mucin-based reporters as well as several fusion proteins containing eight-amino-acid GalNAc-T recognition sequences were glycosylated in this engineered glycocompetent strain of E. coli. The use of one sequence in particular, PPPTSGPT, resulted in site-specific glycan occupancy of approximately 69% at the engineered threonine. The GalNAc present on the purified glycoprotein was oxidized by galactose oxidase and then coupled to hydroxylamine functionalized 20 kDa PEG in the presence of aniline. The glycoprotein could be converted to the PEGylated product at approximately 85% yield and >98% purity as determined by comparison to the products of control reactions.     

Site-specific gene modification by PNAs conjugated to psoralen

DNA-binding molecules, including triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs), can be utilized to introduce site-specific mutations or to promote recombination at selected genomic sites. To further evaluate the utility of PNAs for site-specific gene modification, we tested dimeric bis-PNAs conjugated to psoralen. These PNAs are designed to form a triplex-invasion complex within the supF reporter gene in an episomal shuttle vector and to direct site-specific photoadduct formation by the conjugated psoralen. The psoralen-bis-PNA conjugate was found to direct photoadduct formation to the intended 5'-TpA base step next to the PNA-binding site, and the photoadduct formation efficiency displayed both concentration and UVA irradiation dependence. The effect of PNA-targeted photoadducts in a mammalian system was tested by SV40-based shuttle vector assay. After in vitro binding, we found that photoadducts directed by PNAs conjugated to psoralen-induced mutations at frequencies in the range of 0.46%, 6.5-fold above the background. In a protocol for intracellular gene targeting in the episomal shuttle vector, the psoralen-PNA-induced mutation frequency was 0.13%, 3.5-fold higher than the background. Most of the induced mutations were deletions and single-base-pair substitutions at or adjacent to the targeted PNA-binding and photoadduct-formation sites. When the results are taken together, they demonstrate the ability of bis-PNAs conjugated with psoralen to mediate site-specific gene modification, and they further support the development of PNAs as tools for gene-targeting applications.     

Site-specific modification or rabbit muscle aldolase with fluorescent probes

The site-specific modification of rabbit muscle aldolase A by labeling of thiol residues of Cys-289 with 5-(2-((iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid and Cys-239 with 5-iodoacetamidofluorescein or 4-dimethylamino-phenylazophenyl-4'-maleimide has been described. The method is based on the differences in kinetics of the chemical modification of aldolase thiols with the above reagents either in the presence or in the absence of a competitive inhibitor. The spectral properties of the doubly labeled aldolase derivatives were compared with those of the singly labeled enzyme. The doubly labeled aldolase derivatives exhibited full catalytic activity.     

Site-specific cross-linking of proteins through tyrosine hexahistidine tags

The genetic addition of hexahistidine (H(6)) tags is widely used to isolate recombinant proteins by immobilized metal-affinity chromatography (IMAC). Addition of a tyrosine residue to H(6) tags enabled proteins to be covalently cross-linked under mild conditions in a manner similar to the natural, site-specific cross-linking of tyrosines into dityrosine. A series of seven hexahistidine tags with tyrosines placed in various positions (H(6)Y tags) were added to the amino terminus of the I28 immunoglobulin domain of the human cardiac titin. The H(6)Y-tagged I28 dimerized in the presence of excess Ni(2+) with a K(D) of 200 microM. Treatment of Ni(2+)-dimerized H(6)Y-I28 with an oxidant, monoperoxyphthalic acid (MMPP) or sodium sulfite, resulted in covalent protein multimerization through chelated Ni(2+)-catalyzed cross-linking of the Y residues engineered into the H(6) tag. The protein oligomerization was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The presence of dityrosine in the cross-linked proteins was confirmed by fluorescence emission at 410 nm. Proteins lacking the Y residue in the H(6) tag treated with the same oxidative conditions did not cross-link or exhibit dityrosine fluorescence, despite the presence of an endogenous Y residue. The method may have potential uses in other protein conjugation applications such as protein labeling and interfacial immobilization of proteins on artificial surfaces.     

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.     

Site-specific protein modification to identify the MutL interface of MutH

We have mapped the region for the protein interaction site of the Escherichia coli mismatch repair protein MutH for its activator protein MutL by a site-specific protein modification approach. For this purpose we generated a cysteine-free variant of MutH and 12 variants thereof, each containing a single cysteine residue at surface positions selected on the basis of available structural and sequence information for MutH. All MutH variants displayed wild type activity both in vivo and in vitro. These variants were then site-specifically modified at their cysteine residues with thiol-specific reagents and then tested for their ability to be stimulated in their DNA cleavage activity by the activator protein MutL. Thereby we were able to identify a defined region in the MutH protein that is important for interaction with MutL, and most likely represents the MutL binding site of MutH.     

Site-specific modification and RNA crosslinking of the RNA-binding domain of PKR

RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR is activated in vitro by binding RNA molecules with extensive duplex structure. To further define the nature of the RNA regulation of PKR, we have prepared and characterized site-specifically modified proteins consisting of the PKR 20 kDa RNA-binding domain (RBD). Here we show that the two cysteines found naturally in this domain can be altered by site-directed mutagenesis without loss of RNA binding affinity or the RNA-regulated kinase activity. Introduction of cysteine residues at other sites in the PKR RBD allows for site-specific modification with thiol-selective reagents. PKR RBD mutants reacted selectively with a maleimide to introduce a photoactivatable crosslinking aryl azide at three different positions in the protein. RNA crosslinking efficiency was found to be dependent on the amino acid modified, suggesting differences in access to the RNA from these positions in the protein. One of the amino acid modifications that led to crosslinking of the RNA is located at a residue known to be an autophosphorylation site, suggesting that autophosphorylation at this site could influence the RNA binding properties of PKR. The PKR RBD conjugates described here and other similar reagents prepared via these methods are applicable to future studies of PKR–RNA complexes using techniques such as photocrosslinking, fluorescence resonance energy transfer and affinity cleaving.     

Site-specific covalent attachment of heme proteins on self-assembled monolayers

Naturally occurring hemin cofactor has been functionalized to introduce two terminal alkyne groups. This modified hemin has been successfully covalently attached to mixed self-assembled monolayers of alkanethiols and azide-terminated alkanethiols on gold electrodes using a Cu(I)-catalyzed 1,3-cycloaddition reaction. However these hemin-modified electrodes could not be used to reconstitute apomyoglobin on gold electrodes owing to the hydrophobicity of the alkane thiol self-assembled monolayer. Modification of existing techniques allowed covalent attachment of alkyne-terminated electroactive species onto mixed monolayers of azidothiols and carboxylatoalkanethiols on electrodes using the same Cu(I)-catalyzed 1,3-cycloaddition reaction. Apomyoglobin could be reconstituted using the hemin covalently attached to these hydrophilic electrodes. The electrochemical data, UV-vis absorption data, surface-enhanced resonance Raman spectroscopy data, and atomic force microscopy data indicate the presence of these modified myoglobin proteins on these electrodes. The direct attachment of the heme cofactor of these modified myoglobin proteins to the electrode allows fast electron transfer to the heme center from the electrode and affords efficient O(2)-reducing bioelectrodes under physiological conditions.     

Site-specific labeling of proteins with cyclen-bound transition metal ions

A general procedure for site-specific and reversible labeling of proteins with transition metal ions is described. The method is based on the use of the novel ligand 1-(2-thioethyl)-1,4,7,10-tetraazacyclododecane (TETAC), which specifically and readily reacts with thiol groups. Synthesis of TETAC from 1,4,7,10-tetraazacyclododecane (cyclen) and ethylene disulfide yielded a mixture of products, including TETAC and its oxidized disulfide in 56.4% yield. The procedure for labeling proteins with TETAC is straightforward and led to separation of the TETAC-containing product mixture through gel-filtration chromatography. The resulting protein-TETAC adducts were shown to contain a single TETAC group which bound transition metal ions. Protein-TETACCu²⁺ had a UV-Vis spectrum similar to that of Cu²⁺(cyclen) while the protein-TETACCo²⁺ complex had a different spectrum to that of the cobalt-containing cyclen. This is because attachment to the protein prevented the Co²⁺-containing TETAC from dimerising and binding O₂, which the cobalt-containing cyclen is able to do. The proteins used to develop this labeling procedure were the DNase domain of colicin E9 and its inhibitor protein Im9. Unlike Im9, the DNase does not contain a cysteine residue but the Ser30Cys variant of the DNase was prepared by site-directed mutagenesis. Both Im9 and the Ser30Cys DNase were modified with TETAC and the modifications shown to be structurally and functionally benign through NMR spectroscopy of the modified Im9 and fluorescence spectroscopy binding assays in which DNase-Im9 complexes were formed. The simplicity of the method, and its general application to any protein through the introduction of cysteine by site-directed mutagenesis, suggests it will be of wide use in protein chemistry applications.     

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.     

Site-specific modification of positively-charged surfaces on human serum albumin by malondialdehyde

Malondialdehyde (MDA), a lipid peroxidation product, reacts with lysine residues in proteins. Human serum albumin (HSA) is a major target of MDA-modification of serum proteins. To identify, the modification sites of HSA by MDA in vitro, MDA-treated HSA was digested with a protease and the resulting peptides were subjected to liquid chromatography-tandem mass spectrometry. We identified six peptides, which contained a N-propenal adduct at Lys136, Lys174, Lys240, Lys281, Lys525, and Lys541, and revealed that Lys525 is the most reactive residue for MDA modification. Analysis of electrostatic surface potential of a 3-D model structure of HSA indicates that Lys525 is located at the center of positively charged grooves. The results of this study indicate that the modification of proteins by lipid-derived aldehydes may be influenced by the electrostatic potential of the protein surface.     

Cholesterol modification of proteins

The demonstration over 30 years ago that inhibitors of cholesterol biosynthesis disrupt animal development suggested an intriguing connection between fundamental cellular metabolic processes and the more global processes of embryonic tissue patterning. Adding a new dimension to this relationship is the more recent finding that the Hedgehog family of tissue patterning factors are covalently modified by cholesterol. Here we review the mechanism of the Hedgehog autoprocessing reaction that results in this modification, and compare this reaction to that undergone by other autoprocessing proteins. We also discuss the biological consequences of cholesterol modification, in particular the use of cholesterol as a molecular handle in the spatial deployment of the protein signal in developing tissues. Finally, the developmental consequences of chemical and genetic disruption of cholesterol homeostasis are summarized, along with the potential importance of cholesterol-rich lipid rafts in production of and response to the Hh signal.