Probing homodimer formation of epidermal growth factor receptor by selective crosslinking.

Ligand binding promotes conformational rearrangement of the epidermal growth factor receptor (EGFR) leading to receptor autophosphorylation and downstream signaling. However, transient interactions between unstimulated EGFR molecules on the cell surface are not fully understood. In this report, we describe the investigation of homodimer formation of EGFR by means of an SNAP-tag based selective crosslinking approach (S-CROSS). EGFR homodimers were selectively captured in living cells and utilized for analysis of protein receptor interactions on the plasma membrane and ligand-induced activation. We showed that EGFR forms homodimers in unstimulated cells with efficiencies similar to those seen in cells treated with the epidermal growth factor ligand (EGF) supporting the existence of constitutive transient receptor-receptor interactions. EGFR crosslinked homodimers displayed a substantially increase in kinase activation upon ligand stimulation. Interestingly, in unstimulated cells the levels of spontaneous phosphorylation were found to correlate with the yields of the crosslinked homodimers species. In addition, we demonstrated that this crosslinking approach can be applied to interrogate the effect of small molecule inhibitors on receptor dimerization and kinase activity. Our crosslinking assay provides a new tool to dissect ligand-independent dimerization and activation mechanisms of receptor tyrosine kinases, many of which are important anticancer drug targets.

Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.

Fluorescence in the near-infrared (NIR) spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAP(f) is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG) substrate, leading to covalent attachment of the fluorescent dye to the SNAP(f). This property makes SNAP(f) a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAP(f)-Beta-2 adrenergic receptor (SNAP(f)-ADRß2) fusion protein were created. The ADRß2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAP(f)-ADRß2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAP(f)-ADRß2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAP(f)-ADRß2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAP(f)-ADRß2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging.

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged ß-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.

Site-specific protein labeling by intein-mediated protein ligation.

Intein-mediated protein ligation (IPL) employs an intein to create a protein possessing a C-terminal thioester that can be ligated to a protein or peptide with an amino-terminal cysteine via a native peptide bond. Here we present a procedure to conduct isolation and labeling of recombinant proteins expressed in E. coli using synthetic short peptides possessing a fluorescent moiety. This approach can be readily utilized for site-specific conjugation of a fluorophore to the C-terminus of a protein of interest, without the drawback of non-specific chemical labeling. This chapter also gives a general review of the critical parameters of intein-mediated cleavage and ligation reactions.

Fluorescent site-specific labeling of Escherichia coli expressed proteins with Sfp phosphopantetheinyl transferase.

Fluorescent tagging of proteins has become a critical step in optical analysis of protein function in vitro and in living cells. Here we describe a two-tag system for expression and isolation of a protein of interest from Escherichia coli and subsequent site-specific fluorescent labeling with Sfp phosphopantetheinyl transferase (Sfp synthase). In the example presented, adenoviral protein E3-14.7 K (E14.7) was expressed as a tripartite fusion protein with a fluorophore-targeting peptide tag and a chitin-binding domain. This system allows for rapid isolation of the recombinant fusion protein from crude bacterial cell lysate via a single chitin column. Sfp synthase-mediated labeling with fluorophore conjugated to coenzyme A-SH (CoA-SH) resulted in covalent attachment of a fluorescent dye to a specific residue of the peptide tag via a phosphopantetheinyl linker. The fluorescently labeled E14.7 fusion protein was analyzed with a fluorescence imager and subsequently transfected into mammalian cells for imaging with a fluorescence microscope.

Intein-mediated peptide arrays for epitope mapping and kinase/phosphatase assays.

Synthetic peptides are widely used for production and analysis of antibodies as well as in the study of protein modification enzymes. To circumvent the technical challenges of the existing techniques regarding peptide quantization and normalization, a new method of producing peptide arrays has been developed. This approach utilizes intein-mediated protein ligation that involves linkage of a carrier protein possessing a reactive carboxyl-terminal thioester to a peptide with an amino-terminal cysteine through a native peptide bond. Ligated protein substrates or enzyme-treated samples are arrayed on nitrocellulose membranes with a standard dot-blot apparatus and analyzed by immunoassay. This technique has improved sensitivity and reproducibility, and is suitable for various peptide-based applications. In this report, several experimental procedures including epitope mapping and the study of protein modifications were described.

Reconstitution of glyphosate resistance from a split 5-enolpyruvyl shikimate-3-phosphate synthase gene in Escherichia coli and transgenic tobacco.

A highly N-phosphonomethylglycine (glyphosate)-resistant Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was mapped to identify potential split sites using a transposon-based linker-scanning procedure. Intein-mediated protein complementation was used to reconstitute glyphosate resistance from the genetically divided G2 EPSPS gene in Escherichia coli strain ER2799 and transgenic tobacco.

Preparation of an immunoadsorbent coupled with a recombinant antigen to remove anti-acetylcholine receptor antibodies in abnormal serum.

An immunoadsorbent that removes anti-acetylcholine receptor antibodies (AChRAb) in abnormal serum of myasthenia gravis (MG) patient was efficiently prepared by an expression product, the functional fragment of AChR(alpha205) fused with maltose binding protein (MBP). The ligand can then covalently bind to amylose resin through MBP fusion protein. It was shown from the result of this study with anti-AChR mice sera that the removal rate of AChRAb on this immunoadsorbent reached 87+/-10% (mean value of 10 mice) and the maximally binding capacity of AChRAb was approximately 260 microg/g immunoadsorbent (wet weight). Moreover, the immunoadsorption test of sera in two MG patients indicated that about 90% and 96% of abnormal AChRAb could be eliminated, while other serum components such as albumin, IgG, IgM and IgA only dropped 18%, 35%, 22%, 15% and 24%, 27%, 15%, 12%, respectively, for two MG patient sera. It is anticipated from this study that the immunoadsorbent reported here could, with further development, find its clinical application for removal of AChRAb from patient serum.

Producing peptide arrays for epitope mapping by intein-mediated protein ligation.

Peptide arrays are increasingly used to define antibody epitopes and substrate specificities of protein kinases. Their use is hampered, however, by ineffective and variable binding efficiency of peptides, which often results in low sensitivity and inconsistent results. To overcome these limitations, we have developed a novel method for making arrays of synthetic peptides on various membranes after ligating the peptide substrates to an intein-generated carrier protein. We have conducted screening for optimal carrier proteins by immunoreactivity and direct assessment of binding using a peptide derivatized at a lysine sidechain with fluorescein, CDPEK(fluorescein)DS. Ligation of a synthetic peptide antigen to a carrier protein, HhaI methylase, resulted in an improved retention of peptides and an increased sensitivity of up to 10(4)-fold in immunoassay- and epitope-scanning experiments. Denaturing the ligation products with 2% sodium dodecyl sulfate (SDS) or an organic solvent (20% methanol) prior to arraying did not significantly affect the immunoreactivity of the HhaI methylase-peptide product. Because the carrier protein dominates the binding of ligation products and contains one peptide reactive site, the amount of peptide arrayed onto the membranes can be effectively normalized. This technique was utilized in the alanine scanning of hemagglutinin (HA) antigen using two monoclonal antibodies, resulting in distinguishing the different antigen epitope profiles. Furthermore, we show that this method can be used to characterize the antibodies that recognize phosphorylated peptides. This novel approach allows for synthetic peptides to be uniformly arrayed onto membranes, compatible with a variety of applications.

Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein.

We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this procedure is that each carrier protein molecule ligates only one peptide, ensuring that the ligation product forms a sharp band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrate the effectiveness of this approach by mutational analysis of peptide substrates derived from human cyclin-dependent kinase, Cdc2, which contains a phosphorylation site of human c-Src protein tyrosine kinase.

Soluble expression and affinity purification of functional domain of human acetylcholine receptor alpha-subunit by the modulation of maltose binding protein.

An open reading frame of the alpha-subunit 1-205 residues (alpha205) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR alpha205 as the template and inserted into vector pMAL-c2X. The constructed pMAR alpha205 was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR alpha205 protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR alpha205 could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR alpha205 and AChR alpha205 were similar to that of AChR alpha-subunit from Torpedo.

Generation of an affinity column for antibody purification by intein-mediated protein ligation.

Coupling an antigenic peptide to a solid support is a crucial step in the affinity purification of a peptide-specific antibody. Conventional methods for generating reactive agarose, cellulose or other matrices for peptide conjugation are laborious and can result in a significant amount of chemical waste. In this report, we present a novel method for the facile production of a peptide affinity column by employing intein-mediated protein ligation (IPL) in conjunction with chitin affinity chromatography. A reactive thioester was generated at the C-terminal of the chitin binding domain (CBD) from the chitinase A1 of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein. Peptide epitopes possessing an N-terminal cysteine were ligated to the chitin bound CBD tag. We demonstrate that the resulting peptide columns permit the highly specific and efficient affinity purification of antibodies from animal sera.

Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing.

We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-A resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the N- and C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain Ndelta atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S --> O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.

Protein trans-splicing in transgenic plant chloroplast: reconstruction of herbicide resistance from split genes.

Inteins are intervening protein sequences that undergo self-excision from a precursor protein with concomitant joining of the flanking sequences. Here, we demonstrate intein trans-splicing in Nicotiana tabacum chloroplasts by using the naturally split Ssp DnaE intein. Trans-splicing occurred whether both intein fragments were encoded in the chloroplast or were separated into the chloroplast and nuclear genomes. A biolistic approach was used to integrate two fusion genes, one encoding aminoglycoside-3-adenyltransferase (aadA) and the first 123 aa of the Ssp DnaE intein (In) and the other encoding 36 C-terminal amino acid residues of the Ssp DnaE intein (Ic) and soluble modified green fluorescent protein (smGFP) into N. tabacum plastids. Expression of these gene fragments in the chloroplast resulted in ligated aadA-smGFP due to In-Ic-mediated trans-splicing. Furthermore, an N-terminal portion of the herbicide resistance gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) containing a chloroplast localization signal fused to In (EPSPSn-In) was integrated into the nuclear DNA of N. tabacum by using Agrobacterium tumefaciens-mediated transformation. The remaining EPSPS gene fragment (EPSPSc) fused to Ic (Ic-EPSPSc) was integrated into the chloroplast genome by homologous recombination. Western blot analysis of cell extracts from these plants showed a full-length EPSPS, demonstrating that the EPSPSn-In gene product migrated to the chloroplast and underwent trans-splicing. Furthermore, these transgenic plants displayed improved resistance to the herbicide N-(phosphonomethyl)glycine (glyphosate) when compared with wild-type N. tabacum.