Genetically fused charged peptides induce rapid crystallization of proteins.

We utilized electrostatic interaction to induce rapid crystallization of streptavidin. Simply mixing streptavidins possessing either a positively or negatively charged peptide at their C-terminus generated diffraction-quality crystals in a few hours. We modified the streptavidin crystals with fluorescent molecules using biotin, demonstrating the concept of protein crystals as functional biomaterials.

Open sandwich ELISA: a novel immunoassay based on the interchain interaction of antibody variable region.

We describe an immunoassay that is based on the interchain interaction of separated VL and VH chains from a single chain antibody variable region. In the presence of antigen, the chains reassociate. VL fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 were immobilized on microtiter plates. Samples were coincubated with an M13-displayed VH chain, and assayed with peroxidase-labeled anti-M13 antibody. Signal was detected in direct proportion to the amount of HEL in the sample. Wide dynamic range with < 15 ng/ml sensitivity was attained.

Selective intracellular vaporisation of antibody-conjugated phase-change nano-droplets in vitro.

While chemotherapy is a major mode of cancer therapeutics, its efficacy is limited by systemic toxicities and drug resistance. Recent advances in nanomedicine provide the opportunity to reduce systemic toxicities. However, drug resistance remains a major challenge in cancer treatment research. Here we developed a nanomedicine composed of a phase-change nano-droplet (PCND) and an anti-cancer antibody (9E5), proposing the concept of ultrasound cancer therapy with intracellular vaporisation. PCND is a liquid perfluorocarbon nanoparticle with a liquid-gas phase that is transformable upon exposure to ultrasound. 9E5 is a monoclonal antibody targeting epiregulin (EREG). We found that 9E5-conjugated PCNDs are selectively internalised into targeted cancer cells and kill the cells dynamically by ultrasound-induced intracellular vaporisation. In vitro experiments show that 9E5-conjugated PCND targets 97.8% of high-EREG-expressing cancer cells and kills 57% of those targeted upon exposure to ultrasound. Furthermore, direct observation of the intracellular vaporisation process revealed the significant morphological alterations of cells and the release of intracellular contents.

Crystallization of a photosensitive nitrile hydratase from Rhodococcus sp. N-771.

A photosensitive nitrile hydratase from Rhodococcus sp. N-771 has been crystallized in two different crystal forms in its inactive form. One crystal form belongs to an orthorhombic space group P2(1)2(1)2 with unit cell dimensions of a = 117.4 A, b = 145.7 A and c = 52.1 A, and the other form belongs to a hexagonal space group P6(3)22 with unit cell dimensions of a = 110.2 A and c = 412.1 A.

Post-translational modification is essential for catalytic activity of nitrile hydratase.

Nitrile hydratase from Rhodococcus sp. N-771 is an alphabeta heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, alphaCys112 and alphaCys114 are modified to a cysteine sulfinic acid (Cys-SO2H) and a cysteine sulfenic acid (Cys-SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The alphabeta complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC/MS analysis showed that the anaerobically reconstituted alphabeta complex did not have the modification of alphaCys112-SO2H and aerobic incubation induced the modification. The activity of the reconstituted alphabeta complex correlated with the amount of alphaCys112-SO2H. Furthermore, ESI-LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that alphaCys114 is modified to Cys-SOH together with the sulfinic acid modification of alphaCys112. These results suggest that alphaCys112 and alphaCys114 are spontaneously oxidized to Cys-SO2H and Cys-SOH, respectively, and alphaCys112-SO2H is responsible for the catalytic activity solely or in combination with alphaCys114-SOH.

Light-induced oxidation of iron atoms in a photosensitive nitrile hydratase.

The photoactivation process of a photosensitive nitrile hydratase (NHase) from Rhodococcus sp. N-771 has been investigated by 57Fe Mössbauer spectroscopy and magnetic susceptibility measurements in order to clarify the behavior of iron atoms in the enzyme. Mössbauer spectra of inactive NHase gave two symmetric-doublet components indicating the presence of two iron species, while that of the active NHase gave a single symmetric doublet indicating the presence of a single iron species. Magnetic susceptibility measurements of the inactive and active HNase both showed small effective magnetic moments. These results led us to conclude that one of the two iron atoms incorporated in the NHase is oxidized during photoactivation, namely from a low spin ferrous to a low spin ferric state. This is the first observation of an intramolecular photooxidation phenomena involving iron in a single protein molecule.

Photosensitive nitrile hydratase intrinsically possesses nitric oxide bound to the non-heme iron center: evidence by Fourier transform infrared spectroscopy.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photosensitive enzyme that catalyzes hydration of nitriles to the corresponding amides. Light-induced Fourier transform infrared difference spectra between the inactive and active forms of NHase were measured with both the natural (14N) and 15N-labeled NHases. The results showed, for the first time, that NHase intrinsically possesses nitric oxide (NO) molecules bound to the non-heme iron center. The possible role of NO in the photoactivation process of NHase is discussed.

Open sandwich ELISA with V(H)-/V(L)-alkaline phosphatase fusion proteins.

The Sandwich ELISA is a widely used technique to measure antigen concentration. Recently, a novel ELISA based on the interchain interaction of separated V(H) and V(L) chains from a single antibody variable region (Fv) was proposed (Open Sandwich ELISA). Since it employs a single antibody recognizing one epitope, the assay requires, in essence, only one cycle of incubation and washing steps. To demonstrate this directly, we have constructed a recombinant gene fusion encoding the V(H) chain of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 and Escherichia coli alkaline phosphatase (V(H)-PhoA). The same type of gene fusion using V(L) chain instead of V(H) chain (V(L)-PhoA) was also constructed and the proteins were obtained with an E. coli expression/secretion system. Open Sandwich ELISAs were performed using microtiter plates with immobilized V(L) or V(H) fragment, and V(H)-PhoA or V(L)-PhoA, respectively, as the detection reagent which was simultaneously added to each well with samples. As a result, HEL concentrations in the samples were determined after one round of incubation and washing steps, with a signal generated in a direct relationship to the concentration of HEL added to the reaction mixture. The minimum detectable HEL concentration was approximately 10 ng/ml, which was almost equal to the value previously obtained with plate-immobilized V(L) and V(H) fragment displayed on M13 phage. When the active-site mutant V(H)-PhoA(D101S) was employed instead of V(H)-PhoA and reacted at an optimum pH of 10, a significant enhancement in signal was attained.

Cell-growth control by monomeric antigen: the cell surface expression of lysozyme-specific Ig V-domains fused to truncated Epo receptor.

Previously we have shown that the V(H) and V(L) fragments of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 are weakly associated but can be driven together by antigen. By joining these antibody variable domains to the cytoplasmic portion of the murine erythropoietin receptor, we created a chimeric growth factor receptor that could be activated by HEL. After co-transfection with two plasmids encoding the respective chimeric receptors in IL-3 dependent murine pro-B Ba/F3 cells, a portion of the cells survived under antigen dependent stimulation without IL-3. These surviving cells all showed coexpression of the two chimeric receptor chains and demonstrated HEL dose-dependent growth stimulation without IL-3. When another IL-3 dependent cell line 32D was transfected with a variant of such chimeric receptor with a linker peptide (Gly-Ser-Gly) inserted between V(H)/V(L) and EpoR domains, an improved growth response was attained. These observations suggest the utility of heterodimeric Fv chimeric receptors in creating cells that respond to monomeric antigen.

Expression of a bifunctional chimeric protein A-Vargula hilgendorfii luciferase in mammalian cells.

We have designed and constructed a novel chimeric protein that consisted of a single domain of protein A and luciferase derived from sea-firefly Vargula hilgendorfii with the goal of obtaining a heterofunctional immunological tool. The structural gene of luciferase was fused to the 3' terminus of the D domain gene of protein A with/without a short linker of five amino acids. The resulting constructs under the transcriptional regulation of the Rous sarcoma virus (RSV) promoter, were expressed transiently in simian COS-1 and stably in Chinese hamster ovary (CHO) cells. The properties of the resultant chimeric protein were characterized. The results indicated that the dual properties of the chimeric protein could be retained only after the introduction of a linker of (Gly)4 Ser between the two conjugated moieties. Moreover, the chimeric protein was found to retain at least 50% of the specific activity as compared with the non-fused luciferase. The future prospect of the usage of this chimeric protein in the field of diagnostics was further evaluated by performing bioluminescent immunoassays.

Homogeneous noncompetitive immunoassay based on the energy transfer between fluorolabeled antibody variable domains (open sandwich fluoroimmunoassay).

The antigen-dependent stabilization of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 variable region was monitored with fluorescence resonance energy transfer (FRET) between fluorolabeled heavy chain (VH) and light chain (VL) fragments. The VH and VL fragments labeled with succinimide esters of fluorescein and rhodamine-X, respectively, were mixed in a cooled cuvette, and the change in fluorescence spectra upon antigen addition was monitored. When excited at 490 nm, significant decrease in the fluorescence at 520 nm and its increase at 605 nm were observed when an increasing amount of HEL was added to the mixture in the concentration range of 1-100 micrograms/mL. The assay, named open sandwich fluoroimmunoassay (FIA), is noncompetitive and homogeneous and can be conducted with one clone of antibody. With the use of appropriate antibodies, it is thought to be a quick and inexpensive alternative to the conventional laborious and/or expensive immunoassays.

The inhibition by calmodulin of thyroid-stimulating hormone binding to epididymal fat, testis and thyroid membranes in the guinea-pig.

Calmodulin inhibited 125I-labelled TSH binding to the membranes of various target tissues for TSH (thyroid, epididymal fat and testis) of the guinea-pig. This inhibition was abolished by adding EGTA (1 mmol/l). Calmodulin did not inhibit the binding of 125I-labelled epidermal growth factor (EGF) to these membranes. It is suggested that the inhibitory effect of calmodulin on the binding of TSH to the receptor is specific and that this mechanism is due to the direct binding of calmodulin to receptor membranes. The ability of calmodulin to bind to the membranes was calcium-sensitive while that of TSH was not. The binding of 125I-labelled calmodulin to these membranes increased significantly when the endogenous calmodulin in the membranes was removed by EGTA. It was not inhibited by a pure preparation of TSH, but it was inhibited by contaminated calmodulin in a crude TSH preparation. On the other hand, 125I-labelled TSH binding to these membranes did not change after the removal of endogenous calmodulin. In conclusion, exogenous calmodulin has an inhibitory effect on the binding of TSH but not of EGF to the membranes of guinea-pig thyroid, epididymal fat and testis.

Binding of bovine and porcine pituitary glycoprotein hormone alpha-subunit to TSH antibody in serum of patients with Graves' disease.

The characteristics of autoantibodies reactive with bovine (b) TSH were examined in the sera of six patients with Graves' disease selected on the basis of highly negative values in the TSH receptor assay. Test sera were incubated with other 125I-labeled pituitary glycoprotein hormones and their isolated subunits (alpha and beta) [human (h) TSH, bTSH, porcine (p) TSH, pFSH, bFSH, bLH and equine (e) chorionic gonadotropin (CG)] (purity was confirmed by gel-filtration on Sephadex G-100 and SDS-PAGE), and the antibody bound fraction was precipitated by the addition of anti-human gamma-globulin (goat). Almost all sera showed detectable binding to bTSH, pTSH, pFSH, pTSH-alpha, bFSH-alpha, bLH-alpha, but not to hTSH, hTSH-alpha, hTSH-beta, hFSH, hLH, hCG, pTSH-beta, bLH-beta, eCG-alpha. Exceptions were very low binding to bLH-beta by one serum and to pTSH-beta, by two sera. The level of binding (B/T%) of the patients' sera to pTSH-alpha, bFSH-alpha and bLH-alpha was 3.0-27.7%, 2.6-45.3% and 2.2-39.0%, respectively; that of sera from normal healthy adults was 1.9 +/- 0.3%, 0.8 +/- 0.2% and 0.9 +/- 0.2% (mean +/- SD), respectively. These results indicate that the TSH antibodies recognize mainly an epitope in the alpha subunit of bovine and porcine pituitary glycoprotein hormones (TSH, FSH, LH).

Construction, bacterial expression, and characterization of hapten-specific single-chain Fv and alkaline phosphatase fusion protein.

We have designed and constructed a bacterial expression vector to produce a fusion protein of hapten-specific single-chain Fv (ScFv) and alkaline phosphatase (PhoA) in Escherichia coli. The ScFv gene was assembled using genes encoding the heavy and light chain variable domains of anti-NP (4-hydroxy-3-nitrophenyl acetyl) mouse monoclonal antibody. The ScFv gene was then fused to the 5' terminus of the E. coli PhoA coding region. The expressed fusion protein ScFv(NP)-PhoA was purified using an NP affinity column, and gel-filtration. Characterization of the fusion protein was then performed. The estimated molecular weight by gel filtration was approximately 151 kDa, suggesting the dimerization of the protein. Kinetic constants of ScFv(NP)-PhoA were calculated and compared with those of wild-type PhoA. The k(cat) values of ScFv(NP)-PhoA and wild-type PhoA were 103 (s(-1)) and 96.1 (s(-1)), respectively, showing that PhoA activity was somewhat increased by tethering the molecules. The equilibrium binding constant of ScFv(NP)-PhoA was determined using two different haptens, NP-capronate and NIP(3-iodo-4-hydroxy-5-nitrophenyl acetyl) by means of fluorescence quenching measurements. The obtained binding constants were 2.2 x 10(5) (M-1) for NP-capronate and 1.O x 10(6) (M(-1)) for NIP, respectively. No apparent difference in binding constants was seen between ScFv(NP) and ScFv(NP)-PhoA, showing that sufficient specificity and binding affinity were retained when ScFv(NP) was tethered to alkaline phosphatase. ScFv(NP)-PhoA can be used to detect nanogram concentrations of NP-BSA in ELISA without the use of chemically conjugated secondary antibodies.

Refolding of firefly luciferase immobilized on agarose beads.

The renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation. In this study, firefly luciferase was immobilized on agarose beads at a high concentration. Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution. Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase. These novel observations are described in detail in this paper.

Monitoring of the refolding process for immobilized firefly luciferase with a biosensor based on surface plasmon resonance.

In order to examine the possibility of the use of a surface plasmon resonance (SPR) sensor for real-time monitoring of the process of refolding of immobilized proteins, the refolding of firefly luciferase immobilized on a carboxymethyldextran matrix layer was analyzed. The SPR signal of the immobilized luciferase decreased after unfolding induced by GdnCl and increased gradually in the refolding buffer, while there was no signal change in the reference surface lacking the immobilized protein. The decrease in the SPR signal on unfolding was consistent with the difference between the refractive indices of the native and unfolded protein solutions. The effects of blocking of the excess NHS-groups of the matrix layer on the refolding yield were examined by means of an SPR sensor. The results were consistent with those obtained with the enzymatic activity assay, indicating that the changes in the SPR signal reflected the real-time conformational changes of the immobilized protein. Hence, an SPR biosensor might be used for monitoring of the process of refolding of immobilized proteins and as a novel tool for optimization of the refolding conditions. This is the first demonstration that SPR signal changes reflect the conformational changes of an immobilized protein upon unfolding and refolding.

A growth signal with an artificially induced erythropoietin receptor-gp130 cytoplasmic domain heterodimer.

We report a strategy for generating efficient signal transduction with unnatural heterologous receptor combinations. As previously described [Ueda, H., Kawahara, M. et al. (2000) J. Immunol. Methods 241, 159-170], chimeric receptors composed of the V(H)/V(L) domains of anti-hen egg lysozyme antibody HyHEL-10 and N-terminally truncated erythropoietin receptor (EpoR) can be activated by lysozyme. When the cytoplasmic domains of these receptors were substituted with one derived from gp130, IL-3 dependent Ba/F3 cells expressing both V(H)-gp130 and V(L)-gp130 grew dose-dependently when given lysozyme without IL-3. However, cells expressing the heterologous pair of V(H)-gp130 and V(L)-EpoR also showed more efficient and stricter lysozyme-dependent proliferation in the absence of IL-3, indicating this combination is as an efficient and strict signal transducer as wild-type EpoR. The immunoprecipitation data indicated the existence of a preformed V(H)-gp130 and V(L)-EpoR heterodimer in the absence of lysozyme, suggesting the crucial role of a receptor conformational change in signal triggering as well as wild-type EpoR and gp130. Phosphorylation of JAK2, STAT3, and STAT5 was observed upon the addition of lysozyme, suggesting the activation of both EpoR- and gp130-derived signals.

Truncation of Vargula luciferase still results in retention of luminescence.

Significant amino acid sequence homology in two regions of Vargula hilgendorfii to one in apoaequorin was reported. The intra-amino acid homology in Vargula luciferase between residues 81-312 and 321-540 was 19.3%, and each of this intra-homologous region contained the region homologous to apoaequorin. In order to prove the possibility that only one of the homologous regions is sufficient for luminescence, we have produced a chimeric protein comprising of only the N-terminal homologous region of Vargula luciferase fused to protein A. Comparison of the luminescence of this truncate luciferase indicated that there was 38.5% retention in the bioluminescence of luciferase when compared to that of the mature form of luciferase. This fact may have interesting implications for further study of engineering luciferase.

Construction of green fluorescent protein reporter genes for genotoxicity test (SOS/umu-test) and improvement of mutagen-sensitivity.

Green fluorescent protein (GFP) reporter genes for the bacterial umu-test were constructed. Utilization of tandem, lacUV5 and chimeric trp/umu promoters, and coexpression of the Escherichia coli recA5327 mutant enhanced the GFP expression level fourteen-fold over that of the system with only the umu promoter, thereby improving the sensitivity of the umu-test.

Expanded-bed protein refolding using a solid-phase artificial chaperone.

An efficient solid-phase protein refolding method based on artificial chaperone-assisted refolding is proposed. The method employs insoluble cyclodextrin polymer beads and the expanded-bed technique. Alpha-glucosidase, whose spontaneous refolding yield from a urea-denatured state is up to 30% at a protein concentration of up to 10 microg/ml, could be refolded with a yield that was improved more than two-fold at a protein concentration more than five-fold higher when protein solution was circulated through an expanded bed under optimized conditions. Unlike the conventional liquid-phase artificial system, further steps to purify the refolded product, which are generally needed to remove detergent-cyclodextrin complex and excess cyclodextrin, were unnecessary. In addition, the polymer beads were reusable after simple washing with water, and the continuous system is suitable for easy-scale-up using commercially available devices. This new method is considered to be a powerful means of achieving large-scale protein refolding for industrial protein production.