Sortase-mediated ligations for the site-specific modification of proteins

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Increasing solubility of proteins and peptides by site-specific modification with betaine

Proteins and peptides with low solubility and which aggregate are often encountered in biochemical studies and in pharmaceutical applications of polypeptides. Here, we report a new strategy to improve solubility and prevent aggregation of polypeptides using site-specific modification with the small molecule betaine, which contains a quaternary ammonium moiety. Betaine was site-selectively attached to the N-termini of two aggregation-prone polypeptide models, the bacterial enzyme xanthine-guanine phosphoribosyltransferase (CG-GPRT) and the HIV entry inhibitor peptide CG-T20, utilizing native chemical ligation. N-terminal cysteines for the betaine ligation reactions were generated from His-tagged fusion proteins using TEV protease cleavage. Ligation of the betaine thioester (1) to the N-terminal cysteine-containing polypeptide models proceeded in high yield, though denaturing conditions were required for CG-T20 due to the hydrophobic nature of this peptide. CD spectroscopy and GPRT activity assays indicate that the betaine modification of CG-GPRT and CG-T20 does not significantly affect structure or activity of the polypeptides. Solubility and turbidity measurements of betaine-modified and unmodified polypeptides demonstrate that betaine modification can greatly increase solubility. Finally, it is shown that betaine-modified CG-T20 acts as an inhibitor of the aggregation of unmodified CG-T20.     

A new strategy for the site-specific modification of proteins in vivo

We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-l-phenylalanine into proteins in E. coli. We demonstrate that proteins containing m-acetyl-l-phenylalanine or p-acetyl-l-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells. The labeling reactions are selective and in general proceed with yields of >75%. In specific examples, m-acetyl-l-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes. The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo.     

Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins

Modern drug discovery strongly depends on the availability of target proteins in sufficient amounts and with desired properties. For some applications, proteins have to be produced with specific modifications such as tags for protein purification, fluorescent or radiometric labels for detection, glycosylation and phosphorylation for biological activity, and many more. It is well known that covalent modifications can have adverse effects on the biological activity of some target proteins. It is therefore one of the major challenges in protein chemistry to generate covalent modifications without affecting the biological activity of the target protein. Current procedures for modification mostly rely on non-specific labelling of lysine or cysteine residues on the protein of interest, but alternative approaches dedicated to site-specific protein modification are being developed and might replace most of the commonly used methodologies. In this study, we investigated two novel methods where target proteins can be expressed in E. coli with a fusion partner that allows protein modification in a covalent and highly selective manner. Firstly, we explored a method based on the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) as a fusion tag for site-directed attachment of small molecules. The AGT-tag (SNAP-tag) can accept almost any chemical moiety when it is attached to the guanine base through a benzyl group. In our experiments we were able to label a target protein fused to the AGT-tag with various fluorophores coupled to O6-benzylguanine. Secondly, we tested in vivo and in vitro site-directed biotinylation with two different tags, consisting of either 15 (AviTag) or 72 amino acids (BioEase tag), which serve as a substrate for bacterial biotin ligase birA. When birA protein was co-expressed in E. coli biotin was incorporated almost completely into a model protein which carried these recognition tags at its C-terminus. The same findings were also obtained with in vitro biotinylation assays using pure birA independently over-expressed in E. coli and added to the biotinylation reaction in the test tube. For both biotinylation methods, peptide mapping and LC-MS proved the highly site-specific modification of the corresponding tags. Our results indicate that these novel site-specific labelling reactions work in a highly efficient manner, allow almost quantitative labelling of the target proteins, have no deleterious effect on the biological activity and are easy to perform in standard laboratories.     

Large scale modification of biomolecules using immobilized sortase A from Staphylococcus aureus

Recently, sortase A (SrtA) from Staphyloccus aureus moved into the focus of bioscience because of its ability to incorporate site specific modifications into proteins. The enzyme was mostly used to modify target proteins in an analytical scale, to study biomolecules in their cellular context. In this study, we show the applicability of SrtA mediated ligation for site specific modification of proteins in a large scale. Therefore, the reaction was first optimized using peptides and subsequently new reaction conditions were applied for the large scale biotinylation of interleukin-8. Furthermore, we established C-terminal immobilization of the SrtA on a PEG based resin and could demonstrate maintaining enzymatic activity. Immobilized SrtA significantly facilitates previous ligation protocols as the enzyme can be easily recycled. Also, the removal of excess reaction solution and the whole washing process is significantly accelerated, as centrifugation or filtration techniques can be applied instead of time-consuming chromatography steps.     

Immobilization of proteins to biacore sensor chips using Staphylococcus aureus sortase A

The immobilization of proteins to surfaces is an active area of research due to strong interest in protein-based sensors. Here, we describe a novel method for immobilizing ligand proteins onto Biacore sensor chips using the transpeptidase activity of Staphylococcus aureus sortase A (SrtA). This method provides a robust and gentle approach for the site-directed, covalent coupling of proteins to biosensor chips. Notably, the high specificity of the sortase allows immobilization of proteins from less than pure protein samples allowing short cuts in protein purification protocols.     

Chemoenzymatic methods for site-specific protein modification

In the past decade, numerous chemical technologies have been developed to allow the site-specific post-translational modification of proteins. Traditionally covalent chemical protein modification has been accomplished by the attachment of synthetic groups to nucleophilic amino acids on protein surfaces. These chemistries, however, are rarely sufficiently selective to distinguish one residue within a literal sea of chemical functionality. One solution to this problem is to introduce a unique chemical handle into the target protein that is orthogonal to the remainder of the proteome. In practice, this handle can be a novel peptide sequence, which forms a 'tag' that is selectively and irreversibly modified by enzymes. Furthermore, if the enzymes can tolerate substrate analogs, it becomes possible to engineer chemically modified proteins in a site-specific fashion. This review details the significant progress in creating techniques for the chemoenzymatic generation of protein-small molecule constructs and provides examples of novel applications of these methodologies.     

A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction

A specific mutagenic change in the cDNA of human protein S was introduced by a modification of the polymerase chain reaction that permits the introduction of a mutation at any position in a double-stranded DNA molecule. The method employed four synthetic oligonucleotide primers. One oligonucleotide contained a single-base mismatch to direct the mutagenesis; the other three oligonucleotides were designed to allow selective amplification of the mutated sequence with Thermus aquaticus polymerase. The mutagenized cDNA was cloned into a plasmid vector and transformed into Escherichia coli RR1 cells for characterization. The desired cytosine to guanine change in the target cDNA was confirmed by the predicted appearance of an AluI restriction site and by dideoxynucleotide sequencing. No other sequence changes were detected within the amplified region. This method of site-specific mutagenesis can be applied to any linear double-stranded DNA large enough for primer annealing and obviates specialized cloning vectors, DNA constructs, and selection techniques. It has the advantage over a recently published PCR technique (R. Higuchi, B. Krummel, and R. Saki (1988) Nucleic Acids Res. 16, 7351-7367) in requiring no diafiltration to remove primers between steps and in requiring only a single mutagenic oligonucleotide to be synthesized for each mutant construct made after the initial one.     

New azobenzene derivatives for directed modification of proteins

Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical properties in a free form and after their attachment to the mutant forms of the SsoII restriction endonuclease were studied.     

New immobilization method for immunoaffinity biosensors by using thiolated proteins

A new immobilization method for immunoaffinity (IA) biosensors that ensures the high surface density and the stability of the IA layer was developed. For the immobilization of biomolecules, the molecular recognition protein was first thiolated by covalent conjugation of mercaptopropionic acid, and then the thiolated protein was attached on the gold surface of the transducer. In this work, horseradish peroxidase (HRP) and its antibody were used as a model antigen-antibody, and the following properties of the IA layer prepared by thiolated protein were estimated: (i) biological integrity of HRP after the immobilization process by using activity assay, (ii) charge transfer resistance by immobilization, (iii) mass loading by the surface plasmon resonance (SPR) biosensor, (iv) number of binding sites, and (v) feasibility test for the measurement of capacitive change by the antigen-antibody interaction. Based on these parameters, the immobilization method by using thiolated protein was determined to be feasible for application to IA biosensors.     

An improved method for the covalent coupling of proteins to liposomes

A method is described for the preparation of liposomes carrying covalently attached protein at the outer surface. It is based upon the reaction between proteins, thiolated with the heterobifunctional reagent succinimidyl-S-acetylthioacetate (SATA), and liposomal maleimido-4-(p-phenylbutyryl)phosphatidyl-ethanolamine. Advantages of the procedure are that it is not restricted to proteins carrying native SH-groups and that it is substantially more convenient and less time-consuming than previously published methods. Applications for these liposomes are to be found in the fields of liposome targeting and the production of monoclonal antibodies.     

A spectrophotometric method for quantitation of carboxyl group modification of proteins using Woodward's Reagent K

Reaction of proteins with Woodward's Reagent K in 0.05 ionic strength Tris-HCl, pH 7.8, followed by removal of excess reagent by chromatography on Sephadex G-25 in the same buffer, results in covalently attached chromophores with an absorption maximum at 340 nm and an extinction coefficient of 7000 M-1 cm-1. This absorbance can be used to quantitate the reaction of Woodward's Reagent K with carboxyl groups in proteins, provided sulfhydryl groups do not react. The chromophore also enables specific detection and identification of carboxyl-modified peptides upon separation by chromatography or electrophoresis.     

An enzymatic strategy for site-specific immobilization of functional proteins using microbial transglutaminase

A novel strategy for site-specific immobilization of recombinant proteins was investigated using microbial transglutaminase (MTG). Alkaline phosphatase (AP) was selected as a model protein and tagged with a short peptide (MKHKGS) at the N-terminus to provide a reactive Lys residue for MTG. On the other hand, casein, a well-known substrate for MTG, was chemically attached onto a polyacrylic resin to provide reactive Gln residues for the enzymatic immobilization of the recombinant AP. As a result, we succeeded in MTG-mediated functional immobilization of the recombinant AP onto casein-coated polyacrylic resin. It was found that the immobilized AP prepared using MTG exhibited much higher specific activity than that prepared by chemical modification. Moreover, enzymatic immobilization gave an immobilized formulation with higher stability upon repeated use than that obtained by physical adsorption. Use of this ability of MTG in posttranslational protein modification will provide us with a benign, site-specific immobilization method for functional proteins.     

A self-cleavable sortase fusion for one-step purification of free recombinant proteins

A new protein fusion system has been developed to generate free recombinant protein in a single affinity chromatographic step. The key component in the fusion is the catalytic core of sortase A from Staphylococcus aureus (SrtAc), which recognizes and cleaves the Thr-Gly bond at an LPXTG sequence with moderate activity. The fusion here consists of an N-terminal His6 tag, SrtAc, and an LPETG linker followed by protein of interest at the C-terminus. The fusion protein is expressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography (IMAC). The immobilized fusion then undergoes on-column SrtAc-mediated cleavage at the LPETG site in the presence of Ca2+ and/or triglycine. The target protein with an extra N-terminal glycine is released from the fusion while the N-terminal portion remains bound to the column. Because the cleavage enzyme SrtAc is co-expressed as a fusion with the target protein, the purification system eliminates exogenous proteolysis. This purification approach is simple, robust, inexpensive, time saving, and allows purification of free recombinant protein via one-step chromatography.     

Direct production of proteins with N-terminal cysteine for site-specific conjugation

Proteins with N-terminal cysteine can undergo native chemical ligation and are useful for site-specific N-terminal labeling or protein semisynthesis. Recombinant production of these has usually been by site-specific cleavage of a precursor fusion protein at an internal cysteine residue. Here we describe a simpler route to producing these proteins. Overexpression in E. coli of several proteins containing cysteine as the second amino acid residue yielded products in which the initiating methionine residue had been completely cleaved by endogenous methionine aminopeptidase. While secondary modification of the terminal cysteine was a complicating factor, conditions were identified to eliminate or minimize this problem. Recombinant proteins produced in this way were suitable for site-specific modification of the amino terminus via native chemical ligation technology, as demonstrated by conjugation of a thioester-containing derivative of fluorescein to one such protein. The ability to directly produce proteins with N-terminal cysteine should simplify the application of native chemical ligation technology to recombinant proteins and make the technique more amenable to researchers with limited expertise in protein chemistry.     

Enzyme-mediated site-specific antibody-protein modification using a ZZ domain as a linker

A ZZ domain (ZZ) and alkaline phosphatase (AP), luciferase (Luc), or glucose oxidase (GOD) were conjugated using Sortase A (SrtA) from Staphylococcus aureus. The specific peptidyl linker for SrtA was genetically fused to the C-terminus of ZZ, and the other linker was fused to the N-terminus of AP, Luc, or GOD, respectively. The resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged ZZ and AP, Luc, or GOD were site-specifically conjugated by SrtA through the extra peptidyl linkers in vitro. The SrtA reaction had little influence on either the antibody-binding activity of the ZZ moiety or the enzymatic activity of AP, Luc, or GOD moieties of the conjugates. Additionally, antibody-ZZ-proteins were yielded easily by mixing antibody with ZZ-AP, ZZ-Luc, or ZZ-GOD, allowing their use in an enzyme-linked immunosorbent assay. These results suggest that the enzymatic approach with SrtA facilitates the construction of ZZ-proteins. Furthermore, mixing antibody and ZZ-proteins produces a wide variety of antibody-ZZ-proteins.     

Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by ϕC31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. ϕC31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.     

Interactions of liposomes with serum proteins

The interactions of serum proteins are diverse, complex and can lead to dramatic effects on liposome stability and in vivo behavior; conversely lipids can modify the biological activities of serum proteins. Serum lipoproteins can potentially destabilize bilayer membranes leading to vesicle disruption and loss of contents; irregularities in the lipid bilayer, such as those which exist at phase boundaries, promote the destabilizing effects of lipoproteins. Other serum components such as fibronectin, immunoglobulins and C reactive protein can modify the biological properties of liposomes by promoting interactions with reticuloendothelial cells and/or activation of the complement system. Liposomes can avidly bind certain serum clotting factors, a process which can lead to dramatic effects on the clotting cascade. Thus the interactions of liposomes with serum proteins can reciprocally effect both components involved.