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.     

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

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.     

Recombinant protein hydrazides: application to site-specific protein PEGylation

Here, we describe a novel method for the site-specific C-terminal PEGylation of recombinant proteins. This general approach exploits chemical cleavage of precursor intein-fusion proteins with hydrazine to directly produce recombinant protein hydrazides. This unique functionality within the protein sequence then facilitates site-specific C-terminal modification by hydrazone-forming ligation reactions. This approach was used to generate folded, site-specifically C-terminal PEGylated IFNalpha2b and IFNbeta1b, which retained excellent antiviral activity, demonstrating the utility of this technology in the PEGylation of therapeutic proteins. As this methodology is straightforward to perform, is compatible with disulfide bonds, and is exclusively selective for the protein C-terminus, it shows great potential as general technology for the site-specific engineering and labeling of recombinant proteins.     

Site-specific tetrameric streptavidin-protein conjugation using sortase A

Streptavidin is tetrameric protein which has tight and specific biotin binding affinity, and streptavidin modification of proteins or small molecules is widely used for biotechnology tool. Here, we demonstrate site-specific streptavidin-protein conjugation using enzymes. We focused on sortase A, a transpeptidase from Staphylococcus aureus. A streptavidin-tagged LPETG motif (Stav-LPETG) was expressed in Escherichia coli. We achieved soluble streptavidin expression in E. coli without refolding using a cold shock expression system. Then we successfully conjugated Stav-LPETG with pentaglycine-appended green fluorescence protein (Gly5-GFP) or triglycine-appended glucose oxidase (Gly3-GOD) using sortase A. SDS-PAGE analysis showed site-specific tetrameric streptavidin-protein conjugation with the tagged proteins. In addition, the functions of a Stav-GOD conjugate, i.e., biotin-binding and glucose oxidase activity, were significantly higher compared to those of streptavidin-GOD conjugates prepared by chemical modification.     

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.     

Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro

To extend the (strept)avidin-biotin technology for affinity purification of proteins, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein array without protein purification and many other possible applications, the (strept)avidin-biotin interaction is better when reversible. A gentle enzymatic method to introduce a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins carrying a biotinylation tag has been developed. The optimal condition for efficient in vitro desthiobiotinylation catalyzed by Escherichia coli biotin ligase (BirA) in 1-4h has been established by systematically varying the substrate concentrations, reaction time, and pH. Real desthiobiotinylation in the absence of any significant biotinylation using this enzymatic method was confirmed by mass spectrometric analysis of the desthiobiotinylated tag. This approach was applied to affinity purify desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis to high purity and with good recovery using streptavidin-agarose. The matrix can be regenerated for reuse. This study represents the first successful application of E. coli BirA to incorporate biotin analog to recombinant proteins in a site-specific manner.     

Novel site-specific immobilization of a functional protein using a preferred substrate sequence for transglutaminase 2

Transglutaminase (TGase) catalyzes the formation of a covalent cross-link between a peptide-bound glutamine residue and a lysine residue or primary amine. We have recently identified specific preferred sequences as glutamine-donor substrates in TGase 2 and Factor XIII reactions. By taking advantage of preference of the 12-amino acid sequence for the enzymatic reaction, an efficient immobilization method was established using two different model proteins, glutathione S-transferase (GST) and single-chain fragment antibody (scFv). Both proteins were genetically attached with the preferred substrate sequence to produce a fusion protein. Attachment of the sequence enables the recombinant proteins to act as prominent TGase-substrates and enables them to be immobilized onto chemically amine-terminated gels. Investigation of the biological activities of the two proteins demonstrated their effective immobilization in comparison with that by using a chemically immobilizing method. This established system, which we designated as Transglutaminase-mediated site-specific immobilization method (TRANSIM), would provide site-specific and biologically active conjugation between proteins and several non-protein materials.     

Cloning, protein expression and display of synthetic multi-epitope mycobacterial antigens on Salmonella typhi Ty21a cell surface

Expressing proteins of interest as fusion to proteins of bacterial envelope is a powerful technique for biotechnological and medical applications. The synthetic gene (VacII) encoding for T-cell epitopes of selected genes of Mycobacterium tuberculosis namely, ESAT6, MTP40, 38 kDa, and MPT64 was fused with N- terminus of Pseudomonas syringae ice nucleation protein (INP) outer membrane protein. The fused genes were cloned into a bacterial expression vector pKK223-3. The recombinant protein was purified by Ni-NAT column. VacII gene was displayed on the cell surface of Salmonella typhi Ty21a using N-terminal region of ice nucleation proteins (INP) as an anchoring motif. Glycine method confirmed that VacII was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INP- VacII fusion protein of the expected size (52 kDa).     

Genetic tools for pseudomonads, rhizobia, and other gram-negative bacteria

Gram-negative bacteria are extraordinarily diverse microorganisms that present a wide variety of characteristics worthy of genetic investigation. For historical reasons, the application of recombinant DNA technology to gram-negative bacteria in general has always lagged behind that of E. coli and its close relatives. However, the past 10 years have seen dramatic advances in the development of new tools and vectors for genetic analysis in non-E. coli hosts. Applications include various kinds of genetic manipulation, conjugation, transposition, site-specific recombination, protein secretion, protein purification, cell suicide, microbial ecology, biodegradation, and plant and animal pathogenicity.     

Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

Background

Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling.

Methodology

A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein.

Conclusions

We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.     

Alternative binding proteins: affibody binding proteins developed from a small three-helix bundle scaffold

In recent years, classical antibody-based affinity reagents have been challenged by novel types of binding proteins developed by combinatorial protein engineering principles. One of these classes of binding proteins of non-Ig origin are the so-called affibody binding proteins, functionally selected from libraries of a small (6 kDa), non-cysteine three-helix bundle domain used as a scaffold. During the first 10 years since they were first described, high-affinity affibody binding proteins have been selected towards a large number of targets for use in a variety of applications, such as bioseparation, diagnostics, functional inhibition, viral targeting and in vivo tumor imaging/therapy. The small size offers the possibility to produce functional affibody binding proteins also by chemical synthesis production routes, which has been found to be advantageous for the site-specific introduction of various labels and radionuclide chelators.     

Site-specific chemical protein conjugation using genetically encoded aldehyde tags

We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with α-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.     

Direct protein–protein conjugation by genetically introducing bioorthogonal functional groups into proteins

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein–protein conjugates has gained research interest. In this study, site-specific protein–protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein–protein conjugates and protein complexes of biochemical significance.     

Controlled release of proteins from their poly(ethylene glycol) conjugates: drug delivery systems employing 1,6-elimination

Several tripartate releasable PEG linkers (rPEG) that can provide anchimeric assistance to hydrolysis (cyclization prodrugs) were prepared and, after conjugation to lysozyme demonstrated rapid cleavage in rat plasma compared to nonassisted, permanently bound PEG. By varying the chemical structure and adding steric hindrance, the half-life of the protein conjugates can be adjusted from slow to very fast. The pharmacokinetics (PK) of regeneration of native protein, from various rPEG conjugates can, for the first time, be easily followed in the rat using green fluorescent protein. The PK in mice was also determined for rPEG-Interleukin 2 (rPEG-IL-2) conjugates in vivo using an ELISA assay. Thus, a systematic study of rPEGylated proteins, either in vivo or in vitro during processing, has been investigated based on regeneration of native protein. The employment of releasable PEG polymers substantially broadens the applications of PEGylation drug delivery technology by introducing the benefits of controlled release of native protein therapeutics.     

Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

Background  

Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.     

Site-specific polymer modification of therapeutic proteins

Recent advances in chemoselective ligation technology have made possible the modification of proteins with polymers in a site-specific and controlled manner. These approaches rely on the incorporation of chemoselective anchors into the protein backbone by either chemical or recombinant means, and subsequent modification with a polymer carrying a complementary linker. As a result, the assembly process and the covalent structure of the resulting protein-polymer conjugate are completely controlled, enabling the rational optimization of drug properties, in particular efficacy and pharmacokinetic properties. Application of chemoselective ligation technologies to cytokines and chemokines has led to the generation of new lead proteins for use as erythropoietic agents and HIV fusion inhibitors.     

Introducing genetically encoded aldehydes into proteins

Methods for introducing bioorthogonal functionalities into proteins have become central to protein engineering efforts. Here we describe a method for the site-specific introduction of aldehyde groups into recombinant proteins using the 6-amino-acid consensus sequence recognized by the formylglycine-generating enzyme. This genetically encoded 'aldehyde tag' is no larger than a His(6) tag and can be exploited for numerous protein labeling applications.     

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.     

Site-specific PEGylation at histidine tags

The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.