C-terminal Tag Research Articles

Recombinant protein truncation strategy for inducing bactericidal antibodies to the macrophage infectivity potentiator protein of Neisseria meningitidis and circumventing potential cross-reactivity with human FK506-binding proteins.

A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human proteins, we immunized mice with recombinant truncated type I rMIP proteins that lacked the globular domain and the signal leader peptide (LP) signal sequence (amino acids 1 to 22) and contained the His purification tag at either the N or C terminus (C-term). The immunogenicity of truncated rMIP proteins was compared to that of full (i.e., full-length) rMIP proteins (containing the globular domain) with either an N- or C-terminal His tag and with or without the LP sequence. By comparing the functional murine antibody responses to these various constructs, we determined that C-term His truncated rMIP (-LP) delivered in liposomes induced high levels of antibodies that bound to the surface of wild-type but not Δmip mutant meningococci and showed bactericidal activity against homologous type I MIP (median titers of 128 to 256) and heterologous type II and III (median titers of 256 to 512) strains, thereby providing at least 82% serogroup B strain coverage. In contrast, in constructs lacking the LP, placement of the His tag at the N terminus appeared to abrogate bactericidal activity. The strategy used in this study would obviate any potential concerns regarding the use of MIP antigens for inclusion in bacterial vaccines.

The tmRNA website.

The transfer-messenger RNA (tmRNA) and its partner protein SmpB act together in resolving problems arising when translating bacterial ribosomes reach the end of mRNA with no stop codon. Their genes have been found in nearly all bacterial genomes and in some organelles. The tmRNA Website serves tmRNA sequences, alignments and feature annotations, and has recently moved to http://bioinformatics.sandia.gov/tmrna/. New features include software used to find the sequences, an update raising the number of unique tmRNA sequences from 492 to 1716, and a database of SmpB sequences which are served along with the tmRNA sequence from the same organism.

Polylysine-mediated translocation of the diphtheria toxin catalytic domain through the anthrax protective antigen pore.

The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores in the endosomal membrane, which translocate the effector proteins of the toxin to the cytosol. Effector proteins bind to oligomeric PA via their respective N-terminal domains and undergo N- to C-terminal translocation through the pore. Earlier we reported that a tract of basic amino acids fused to the N-terminus of an unrelated effector protein (the catalytic domain diphtheria toxin, DTA) potentiated that protein to undergo weak PA-dependent translocation. In this study, we varied the location of the tract (N-terminal or C-terminal) and the length of a poly-Lys tract fused to DTA and examined the effects of these variations on PA-dependent translocation into cells and across planar bilayers in vitro. Entry into cells was most efficient with ∼12 Lys residues (K12) fused to the N-terminus but also occurred, albeit 10–100-fold less efficiently, with a C-terminal tract of the same length. Similarly, K12 tracts at either terminus occluded PA pores in planar bilayers, and occlusion was more efficient with the N-terminal tag. We used biotin-labeled K12 constructs in conjunction with streptavidin to show that a biotinyl-K12 tag at either terminus is transiently exposed to the trans compartment of planar bilayers at 20 mV; this partial translocation in vitro was more efficient with an N-terminal tag than a C-terminal tag. Significantly, our studies with polycationic tracts fused to the N- and C-termini of DTA suggest that PA-mediated translocation can occur not only in the N to C direction but also in the C to N direction.

Cloning, expression, purification, crystallization and preliminary X-ray characterization of allantoinase from Bacillus licheniformis ATCC 14580.

Allantoinase, a member of the amidohydrolase superfamily, exists in a wide variety of organisms, including bacteria, fungi, plants and a few animals, such as fishes and amphibians. Allantoinase catalyzes the reversible hydrolysis of allantoin into allantoate by hydrolytic cleavage of the N1-C2 amide bond of the five-membered hydantoin ring. Allantoinase from Bacillus licheniformis (AllBali) presents an inverted enantioselectivity towards allantoin (R-enantioselective), which is a distinguishable feature that is not observed for other allantoinases. In this work, B. licheniformis ATCC 14580 allantoinase (AllBali) containing a C-terminal His6 tag was overproduced in Escherichia coli and purified to homogeneity. Crystals of AllBali were obtained by the vapour-diffusion method using 0.1 M potassium thiocyanate, 20%(w/v) polyethylene glycol 3350 as a crystallization solution. X-ray diffraction data were collected to a resolution of 3.5 Å with an Rmerge of 29.2% from a crystal belonging to space group P12₁1, with unit-cell parameters a=54.93, b=164.74, c=106.89 Å, β=98.49°. There are four molecules in the asymmetric unit with a solvent content of 47% as estimated from the Matthews coefficient (VM=2.34 Å3 Da(-1)).

In situ proteolysis, crystallization and preliminary X-ray diffraction analysis of a VHH that binds listeria internalin B.

The variable region of camelid heavy-chain antibodies produces the smallest known antibody fragment with antigen-binding capability (a VHH). The VHH R303 binds internalin B (InlB), a virulence factor expressed by the pathogen Listeria monocytogenes. InlB is critical for initiation of Listeria infection, as it binds a receptor (c-Met) on epithelial cells, triggering the entry of bacteria into host cells. InlB is surface-exposed and is required for virulence, hence a VHH targeting InlB has potential applications for pathogen detection or therapeutic intervention. Here, the expression, purification, crystallization and X-ray diffraction of R303 are reported. Crystals of R303 were obtained following in situ proteolysis with trypsin. Gel filtration and SDS-PAGE revealed that trypsin removed the C-terminal tag region of R303, facilitating crystal formation. Crystals of R303 diffracted to 1.3 Å resolution and belonged to the monoclinic space group P2₁, with unit-cell parameters a=46.4, b=31.2, c=74.8 Å, β=93.8°. The crystals exhibited a Matthews coefficient of 1.95 Å3 Da(-1) with two molecules in the asymmetric unit.

An optimized intein-mediated protein ligation approach for the efficient cyclization of cysteine-rich proteins.

Head-to-tail backbone cyclization of proteins is a widely used approach for the improvement of protein stability. One way to obtain cyclic proteins via recombinant expression makes use of engineered Intein tags, which are self-cleaving protein domains. In this approach, pH-induced self-cleavage of the N-terminal Intein tag generates an N-terminal cysteine residue at the target protein, which then attacks in an intramolecular reaction the C-terminal thioester formed by the second C-terminal Intein tag resulting in the release of the cyclic target protein. In the current work we aimed to produce a cyclic analog of the small γ-Ec-1 domain of the wheat metallothionein, which contains six cysteine residues. During the purification process we faced several challenges, among them premature cleavage of one or the other Intein tag resulting in decreasing yields and contamination with linear species. To improve efficiency of the system we applied a number of optimizations such as the introduction of a Tobacco etch virus cleavage site and an additional poly-histidine tag. Our efforts resulted in the production of a cyclic protein in moderate yields without any contamination with linear protein species.

Improvement of a Sulfolobus-E. coli shuttle vector for heterologous gene expression in Sulfolobus acidocaldarius.

A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

Duplication of Genes in an ATP-binding Cassette Transport System Increases Dynamic Range While Maintaining Ligand Specificity

Many bacteria exist in a state of feast or famine where high nutrient availability leads to periods of growth followed by nutrient scarcity and growth stagnation. To adapt to the constantly changing nutrient flux, metabolite acquisition systems must be able to function over a broad range. This, however, creates difficulties as nutrient concentrations vary over many orders of magnitude, requiring metabolite acquisition systems to simultaneously balance ligand specificity and the dynamic range in which a response to a metabolite is elicited. Here we present how a gene duplication of a periplasmic binding protein in a mannose ATP-binding cassette transport system potentially resolves this dilemma through gene functionalization. Determination of ligand binding affinities and specificities of the gene duplicates with fluorescence and circular dichroism demonstrates that although the binding specificity is maintained the Kd values for the same ligand differ over three orders of magnitude. These results suggest that this metabolite acquisition system can transport ligand at both low and high environmental concentrations while preventing saturation with related and less preferentially metabolized compounds. The x-ray crystal structures of the β-mannose-bound proteins help clarify the structural basis of gene functionalization and reveal that affinity and specificity are potentially encoded in different regions of the binding site. These studies suggest a possible functional role and adaptive advantage for the presence of two periplasmic-binding proteins in ATP-binding cassette transport systems and a way bacteria can adapt to varying nutrient flux through functionalization of gene duplicates.

Saccharomyces cerevisiae 표면 발현을 이용한 붉바리 신경괴사 바이러스 외피단백질의 생산

The studies of marine viruses in terms of viral isolation and detection have been limited due to the high mutation rate and genetic diversity of marine viruses. Of the modern methods currently used to detect marine viruses, serological methods based on enzyme-linked immunosorbent assay (ELISA) are the most common. They depend largely on the quality of the antibodies and on highly purified suitable antigens. Recently, a new experimental system for using viral capsid protein as an antigen has been developed using the yeast surface display (YSD) technique. In the present study, the capsid protein gene of the red-spotted grouper nervous necrosis virus (RGNNV) was expressed and purified via YSD and HA-tagging systems, respectively. Two regions of the RGNNV capsid protein gene, RGNNV1 and RGNNV2, were individually synthesized and subcloned into a yeast expression vector, pCTCON. The expressions of each RGNNV capsid protein in the Saccharomyces cerevisiae strain EBY100 were indirectly detected by flow cytometry with fluorescently labeled antibodies, while recognizing the C-terminal c-myc tags encoded by the display vector. The expressed RGNNV capsid proteins were isolated from the yeast surface through the cleavage of the disulfide bond between the Aga1 and Aga2 proteins after β-mercaptoethanol treatment, and they were directly detected by Western blot using anti-HA antibody. These results indicated that YSD and HA-tagging systems could be applicable to the expressions and purification of recombinant RGNNV capsid proteins.

Abstract 881: Identification of autoantibody biomarkers to wild-type and mutant p53 in pancreatic and ovarian cancer

Abstract Introduction: Mutations in TP53 are common in early pancreatic and ovarian cancer development, and are detected in the majority of pancreatic cancers (75%) and serous ovarian cancers (97%). Mutations in TP53 induce autoantibody (AAb) immune responses, which may be biomarkers for the early detection of these cancers. The most common mutations in p53 occur in the DNA binding domain at residues that directly contact the DNA (Arg175, 248, and 273). In contrast, preliminary studies suggest that mutations in the N- and C- termini of p53 alter the thermodynamic stability of p53, leading to increased accumulation in tumors. It is not known if the location of the mutations impacts immunogenicity, or if p53-AAb are specific to mutant p53 proteins. Methods: To evaluate AAb to mutant p53 proteins, we generated custom programmable protein microarrays displaying 52 common p53 point mutations. cDNAs encoding full-length p53 with c-terminal GST tags were printed on arrays with anti-GST antibody, and transcribed and translated in situ using HeLa expression lysate. The microarrays were screened with sera from pancreatic cancer cases (Stage I-III, n=60), healthy and benign disease controls (n=63), ovarian cancer cases (n=19) and controls (n=17). Bound IgG was detected by fluorescence. For validation, four immunogenic mutant p53 proteins Arg248Met, Pro278Ser, Arg213Leu, Gly187Ser and wild-type p53 were individually displayed in 96-well format using a programmable ELISA. The ELISAs were re-screened with a subset of the sera from pancreatic cancer cases (n=49) and controls (n=47) and all of the ovarian cancer cases and controls. Cutoff values were established to determine if a patient is positive for all wild-type and mutant p53 AAb by measuring the mean antigen florescence intensity +2 standard deviations of the controls. Results: AAb were detected to all four mutant and wild-type p53 proteins in sera from 8/49 (16%) of pancreatic cases and 2/47 (4.2%) of pancreatic controls, but the results were not statistically significant. In comparison, AAb to all four mutant and wild-type p53 proteins were identified in sera from 8/19 (42%) of ovarian cases and 0/17 (0%) of healthy control women (p ≤ 0.005). All sera with AAb to mutant p53 also had measurable AAb to wild-type p53 protein. All patients with immune responses had evidence of broad-reactivity to at least at least two mutant p53 proteins, as well as to wild-type p53. Conclusion: Our results indicate that patients with serum AAb to wild type p53 protein also have AAb to multiple mutant p53 antigens, suggesting degeneracy of the epitope specificity. No patients have been identified with AAb specific to mutant-only p53. We observed inter-patient variation in the patterns of immune reactivity to p53 mutations that is of unclear significance. Correlation with tumor p53 mutation status is ongoing. Citation Format: Benjamin A. Katchman, Rizwan Alam, Garrick Wallstrom, Joshua LaBaer, Michael A. Hollingsworth, Daniel W. Cramer, Karen S. Anderson. Identification of autoantibody biomarkers to wild-type and mutant p53 in pancreatic and ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 881. doi:10.1158/1538-7445.AM2014-881

Searching for an ideal vaccine candidate among different MERS coronavirus receptor-binding fragments—The importance of immunofocusing in subunit vaccine design

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is currently spreading among humans, making development of effective MERS vaccines a high priority. A defined receptor-binding domain (RBD) in MERS-CoV spike protein can potentially serve as a subunit vaccine candidate against MERS-CoV infections. To identify an ideal vaccine candidate, we have constructed five different versions of RBD fragments, S350-588-Fc, S358-588-Fc, S367-588-Fc, S367-606-Fc, and S377-588-Fc (their names indicate their residue range in the spike protein and their C-terminal Fc tag), and further investigated their receptor binding affinity, antigenicity, immunogenicity, and neutralizing potential. The results showed that S377-588-Fc is among the RBD fragments that demonstrated the highest DPP4-binding affinity and induced the highest-titer IgG antibodies in mice. In addition, S377-588-Fc elicited higher-titer neutralizing antibodies than all the other RBD fragments in mice, and also induced high-titer neutralizing antibodies in immunized rabbits. Structural analysis suggests that S377-588-Fc contains the stably folded RBD structure, the full receptor-binding site, and major neutralizing epitopes, such that additional structures to this fragment introduce non-neutralizing epitopes and may also alter the tertiary structure of the RBD. Taken together, our data suggest that the RBD fragment encompassing spike residues 377-588 is a critical neutralizing receptor-binding fragment and an ideal candidate for development of effective MERS vaccines, and that adding non-neutralizing structures to this RBD fragment diminishes its neutralizing potential. Therefore, in viral vaccine design, it is important to identify the most stable and neutralizing viral RBD fragment, while eliminating unnecessary and non-neutralizing structures, as a means of “immunofocusing”.

Francisella novicida pathogenicity island encoded proteins were secreted during infection of macrophage-like cells.

Intracellular pathogens and other organisms have evolved mechanisms to exploit host cells for their life cycles. Virulence genes of some intracellular bacteria responsible for these mechanisms are located in pathogenicity islands, such as secretion systems that secrete effector proteins. The Francisella pathogenicity island is required for phagosomal escape, intracellular replication, evasion of host immune responses, virulence, and encodes a type 6 secretion system. We hypothesize that some Francisella novicida pathogenicity island proteins are secreted during infection of host cells. To test this hypothesis, expression plasmids for all Francisella novicida FPI-encoded proteins with C-terminal and N-terminal epitope FLAG tags were developed. These plasmids expressed their respective epitope FLAG-tagged proteins at their predicted molecular weights. J774 murine macrophage-like cells were infected with Francisella novicida containing these plasmids. The FPI proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of the respective genes that were deficient for intramacrophage growth. Using these expression plasmids, the localization of the Francisella pathogenicity island proteins were examined via immuno-fluorescence microscopy within infected macrophage-like cells. Several Francisella pathogenicity island encoded proteins (IglABCDEFGHIJ, PdpACE, DotU and VgrG) were detected extracellularly and they were co-localized with the bacteria, while PdpBD and Anmk were not detected and thus remained inside bacteria. Proteins that were co-localized with bacteria had different patterns of localization. The localization of IglC was dependent on the type 6 secretion system. This suggests that some Francisella pathogenicity island proteins were secreted while others remain within the bacterium during infection of host cells as structural components of the secretion system and were necessary for secretion.

Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti

Mesorhizobium loti MAFF303099, a nitrogen-fixing symbiotic bacterium, harbors degradation pathway I for pyridoxine (PN); a free form of vitamin B6. Pyridoxine 4-oxidase (PNOX), a monomeric glucose-methanol-choline (GMC) oxidoreductase family enzyme, is the first enzyme in the pathway. It catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal. PNOX with a C-terminal His6 tag was overexpressed in E.coli JM109 cells and purified with a Ni-NTA agarose column and a QA52 column. The tertiary structures of PNOX and a complex of PNOX with pyridoxamine (PM), which is a substrate analog, were determined at 2.2 Å and at 2.1 Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. The FAD interacts with the PNOX protein through a network of hydrogen bonds, which are mainly found in the ribose and pyrophosphate moieties of the FAD molecule. The surface structure of PNOX molecule showed that it had an opening socket for access of substrates. The opening was followed by a tunnel that was linked to the active site cavity. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 Å from the N4′ atom of PM. The activities of H460A and H462A mutant PNOXs were very low, and H460A/H462A double mutant PNOX exhibited no activity. His462 may act as a general base for abstraction of a proton from the 4′-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4′ atom in PM is located at 3.2 Å, and the hydride ion from the C4′ atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase family shows that Pro504 in PNOX corresponds to Asn or His of the conserved His-Asn or His-His pair in other GMC oxidoreductases.

Improved soluble expression of a single-chain antibody fragment in E. coli for targeting CA125 in epithelial ovarian cancer

Production of antibody fragments in heterologous hosts such as Escherichia coli provides a unique and cost-effective method to develop engineered vectors for tumor targeting. A single-chain Fragment variable (scFv) of the murine monoclonal antibody MAb-B43.13 targeting CA125 in epithelial ovarian cancer was previously developed, expressed, purified and proposed as a functional targeting entity for biomedical applications. However, the yields from its soluble expression in heterologous systems were very low for any practical use in preclinical translational research; leave alone the defeated objective of convenient and cost-effective production. In the present work, the anti-CA125 scFv gene was re-organized and sub-cloned into pET-22b(+) vector to be in frame with the pelB leader peptide for periplasmic localization and C-terminal hexa-histidine tag to facilitate downstream purification. Six variants of the scFv were constructed to investigate the impact of variable domain orientations, inter-domain peptide linker sequences and codon optimization on the soluble expression of the scFv using Rosetta 2(DE3) as the E. coli host supplemented with tRNAs for rare codons. Expression in shake flask cultures under the control of an inducible T7 promoter and subsequent purification by cobalt based immobilized metal affinity chromatography yielded differential amounts of high purity scFv for all constructs. Here, we report up to 14-fold increase in the soluble expression of the scFv primarily as a result of codon optimization with minor effects from inter-domain peptide linkers and variable domain orientation in the anti-CA125 scFv molecule. All the scFv constructs expressed and purified were found to be immunoreactive for in vitro targeting of CA125 antigen.

Preliminary X-ray crystallographic studies of the TIR domain of human Toll-like receptor 6.

Toll-like receptor (TLR) proteins have been identified and shown to play a role in the innate immune response. TLR6 associated with TLR2 can recognize diacylated lipoprotein. In this study, the human TLR6 TIR domain corresponding to amino acids 640-796 was overexpressed in Escherichia coli using engineered C-terminal His tags. The TLR6 TIR domain was then purified to homogeneity and crystallized at 20°C. Finally, X-ray diffraction data were collected to a resolution of 2.2 Å from a crystal belonging to space group C2, with unit-cell parameters a = 127.60, b = 44.20, c = 75.72 Å, β = 118.89°

Molecular regulation of lysophosphatidic acid receptor 1 trafficking to the cell surface

The lysophosphatidic acid receptor 1 (LPA1), a G-protein coupled receptor, regulates cell proliferation, migration, and cytokine release. Here, we investigate the molecular signature of LPA1 trafficking to the cell surface. The overexpressed LPA1 with a C-terminal V5 tag (LPA1-V5) is majorly expressed on the cell surface, while two deletion mutants (C320 and ∆84–87) failed to be trafficked to the cell surface. Further, site-directed mutagenesis analysis of the LPA1 revealed that Ile325, Tyr85, and Leu87 within these two fragments regulate LPA1 maturation and trafficking to the cell surface. Over-expression of Sar1, a component of coat protein complex II (COPII), enhances glycosylation of LPA1 wild type, but not these mutants. The mutants of LPA1 are majorly localized in the endoplasmic reticulum (ER) and exhibit a higher binding affinity to heat shock protein 70 (Hsp70), when compared to the LPA1 wild type. Further, we found that all these mutants failed to increase phosphorylation of Erk, and the cytokine release in response to LPA treatment. These results suggest that Ile325, Tyr85, and Leu87 within LPA1 are essential for LPA1 protein properly folding in the ER.

Long-range effects of tag sequence on marginally stabilized structure in HIV-1 p24 capsid protein monitored using NMR

N-terminal domain of HIV-1 p24 capsid protein is a globular fold composed of seven helices and two β-strands with a flexible structure including the α4–5 loop and both N- and C-terminal ends. However, the protein shows a high tendency (48%) for an intrinsically disordered structure based on the PONDR VL-XT prediction from the primary sequence. To assess the possibility of marginally stabilized structure under physiological conditions, the N-terminal domain of p24 was destabilized by the addition of an artificial flexible tag to either N- or C-terminal ends, and it was analyzed using T1, T2, hetero-nuclear NOE, and amide-proton exchange experiments. When the C-terminal tag (12 residues) was attached, the regions of the α3–4 loop and helix 6 as well as the α4–5 loop attained the flexible structures. Furthermore, in the protein containing the N-terminal tag (27 residues), helix 4 in addition to the above-mentioned area including α3–4 and α4–5 loops as well as helix 6 exhibited highly disordered structures. Thus, the long-range effects of the existence of tag sequence was observed in the stepwise manner of the appearance of disordered structures (step 1: α4–5 loop, step 2: α3–4 loop and helix 6, and step 3: helix 4). Furthermore, the disordered regions in tagged proteins were consistent with the PONDR VL-XT disordered prediction. The dynamic structure located in the middle part (α3–4 loop to helix 6) of the protein shown in this study may be related to the assembly of the viral particle.

In situ protein microarrays capable of real-time kinetics analysis based on surface plasmon resonance imaging

In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein–protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (∼3–7×10−13M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein–protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields.

Production and applications of an N-terminally-truncated recombinant beta-haemolysin from Staphylococcus aureus

The beta-haemolysin of Staphylococcus aureus (SA-hlb) is a secreted neutral sphingomyelinase (nSMase) implicated in the pathogenesis of infection and responsible for the characteristic in vitro ‘hot–cold’ haemolytic ability of the bacterium. Here, we describe the production of a biologically active N-terminally-truncated recombinant SA-hlb protein for use in in vitro assays and as a research tool. Using local isolates of S. aureus, we PCR-amplified an SA-hlb DNA sequence of 891 nucleotides, 99 nucleotides shorter than the full-length molecule, before cloning and sequencing (GenBank accession no. JN580071). The pQE.TriSystem vector (Qiagen, Germany) was used to express recombinant SA-hlb (r-SA-hlb) with a C-terminal 8xHis tag in Escherichia coli JM107 cells. Both JM107 lysate and the purified r-SA-hlb possessed hot–cold lytic activity against sheep and buffalo erythrocytes, which was abolished by incubation at ≥90 °C for 30 min or exposure to dithiothreitol, and could be neutralized by bovine immune sera. Purified r-SA-hlb was also cytotoxic to buffalo mononuclear cells and was effective as a coating antigen for indirect ELISA to screen for reactive sera. Importantly, the r-SA-hlb was suitable for use as a β-toxin in the modified CAMP test. We conclude that the r-SA-hlb protein produced was functionally active and has numerous potential applications.

Resonance assignment and secondary structure determination of full length human Dickkopf 4 (hDkk4), a secreted, disulphide-rich Wnt inhibitor protein

A number of proteins have been shown to modulate canonical Wnt signalling at the cell surface, including members of the Dickkopf (Dkk) family (Baron and Rawadi in J Endocrinol 148:2635-2643, 2007; Cruciat and Niehrs in Cold Spring Harb Perspect Biol 5:a015081, 2013). The Dkk family includes four secreted proteins (Dkk1-4), which are characterised by two highly conserved cysteine-rich regions corresponding to C24-C73 and C128-C201 in human Dkk4 (hDkk4). Here we report essentially complete backbone and comprehensive side chain (15)N, (13)C and (1)H NMR assignments for full length mature hDkk4 (M1-L207) containing a short C-terminal hexa-histidine tag (E208-H222). Analysis of the backbone chemical shift data obtained indicates that there is a very limited amount of regular secondary structure, with only small stretches of β-strand identified in both cysteine-rich regions. The N-terminal region of hDkk4 (M1-G21) and the relatively long linker between the two cysteine-rich regions (E77-Q123) appear to be unstructured and relatively mobile.