Bioluminescence immunoassay for cortisol using recombinant aequorin as a label

The analysis of hormones in saliva is a powerful tool in the assessment of a patient's endocrine function, since it allows multiple noninvasive samplings. Since salivary levels of most hormones are 10 to 50 times lower than plasma levels, accurate and highly sensitive assays are needed for saliva measurements. Herein, we describe the development of a solid-phase competitive immunoassay for cortisol in saliva, in which a mutant of the photoprotein aequorin has been used as a label. We have chemically conjugated cortisol to aequorin at different molar ratios. The various cortisol-aequorin conjugates were characterized in terms of bioluminescent activity and affinity for the anti-cortisol antibody. The conjugate that gave the best analytical performance was used for the development of the immunoassay and the analysis of cortisol in saliva samples. The conjugates were stable for at least 6 months when stored at 4 degrees C. The method fulfilled all the standard requirements of precision and accuracy. The optimized immunoassay gave a detection limit of 300 fmol/tube, corresponding to 3 nmol/L, with a linear dynamic range of 10-1000 nmol/L. Therefore, cortisol can be detected down to 0.1 ng in 100 microl of saliva sample using this assay, without any sample pretreatment. This detection limit is almost one order of magnitude lower than the physiological levels of salivary cortisol, which are reported to be 10-25 nmol/L. This allows the quantification of salivary cortisol to be performed in the linear range of the calibration curve, which is most reliable for quantification purposes.     

Bioluminescence detection of proteolytic bond cleavage by using recombinant aequorin

Detection of proteolytic bond cleavage was achieved by taking advantage of the bioluminescence emission generated by the photoprotein aequorin. A genetically engineered HIV-1 protease substrate was coupled with a cysteine-free mutant of aequorin by employing the polymerase chain reaction to produce a fusion protein that incorporates an optimum natural protease cleavage site. The fusion protein was immobilized on a solid phase and employed as the substrate for the HIV-1 protease. Proteolytic bond cleavage was detected by a decrease in the bioluminescence generated by the aequorin fusion protein on the solid phase. A dose-response curve for HIV-1 protease was constructed by relating the decrease in bioluminescence signal with varying amounts of the protease. The system was also used to evaluate two competitive and one noncompetitive inhibitor of the HIV-1 protease. Among the advantages of this assay is that by using recombinant methods a complete bioluminescently labeled protease recognition site can be designed and produced. The assay yields very sensitive detection limits, which are inherent to bioluminescence-based methods. An application of this system may be in the high-throughput screening of biopharmaceutical drugs that are potential inhibitors of a target protease.     

Bioluminescence of the Ca-binding photoprotein, aequorin, after histidine modification

Modification studies of the 5 histidine residues in aequorin employing site-directed mutagenesis and diethyl pyrocarbonate suggested that His¹⁶⁹ may be the site of binding of molecular oxygen in aequorin. The modification of this residue led to complete loss of activity, whereas modification of the remaining 4 histidine residues yielded mutant aequorins with varying bioluminescence activities.     

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.     

Adapter protein for site-specific conjugation of payloads for targeted drug delivery

High-affinity interactions of two fragments of human RNase I (1-15-aa Hu-tag and 21-125-aa HuS adapter protein) can be used for assembly of targeting drug delivery complexes. In this approach, a targeting protein is expressed as a fusion protein with a 15-aa Hu-tag, while HuS is conjugated to a drug (or a drug carrier) creating a "payload" module, which is then bound noncovalently to the Hu-tag of the targeting protein. Although this approach eliminates chemical modifications of targeting proteins, the payload modules are still constructed by random cross-linking of drugs or drug carriers to an adapter protein that might lead to functional heterogeneity of the complexes. To avoid this problem, we engineered an adapter protein HuS(N88C) with an unpaired cysteine in position 88 that can be directly modified without interference with activity of assembled targeting complexes. HuS(N88C) binds Hu-tagged annexin V with K(D) of 50 +/- 6 nM, which is comparable to that of wild-type HuS. To demonstrate the utility of HuS(N88C) for developing uniform payload modules, we constructed a HuS(N88C)-lipid conjugate and inserted it into preformed liposomes loaded with a fluorescent dye. Targeting proteins, Hu-tagged vascular endothelial growth factor or Hu-tagged annexin V, were docked to liposomes decorated with HuS, and the assembled complexes delivered liposomes selectively to target cells.     

Screening of PC and PMMA-binding peptides for site-specific immobilization of proteins

In the present study, we used proteomic research technology to develop a method for the screening and evaluation of material-binding peptides for protein immobilization. Using this screening method, soluble Escherichia coli proteins that preferentially adsorbed onto polycarbonate (PC) and poly(methylmethacrylate) (PMMA) as model plastic materials were first isolated and identified by 2-dimensional electrophoresis (2DE) combined with peptide mass fingerprinting (PMF). The genes of identified protein candidates (ELN, MLT, OMP, and BIF) that exhibited a hexahistidine tag (6×His-tag) were over-expressed by E. coli BL21 (DE3), and the proteins were purified by IMAC affinity chromatography. The candidates for PC and PMMA-binding peptides were isolated from peptide fragments from affinity protein candidates, which were digested with trypsin and chymotrypsin. Consequently, 5 candidates for the PC-binding peptide and 2 candidates for the PMMA-binding peptide were successfully identified by MALDI-TOF MS. All of the peptides identified were introduced to the C-terminus of glutathione S-transferase (GST) as a model protein for immobilization. Adsorption of peptide-fused and wild-type GSTs onto the plastic surfaces was directly monitored using a quartz crystal microbalance (QCM) device. Consequently, genetic fusion of PC-MLT8 and PC-OMP6 as PC-binders and PM-OMP25 as a PMMA-binder significantly enhanced the adsorption rates of GST, achieving an adsorption density that was more than 10 times higher than that of wild-type GST. Furthermore, the residual activity levels of GST-PC-OMP6 and GST-PM-OMP25 in the adsorption state were 2 times higher than that of wild-type GST. Thus, the PC and PMMA-binding peptides identified in this study, namely PC-OMP6 and PM-OMP25, were considerably useful for site-specific immobilization of proteins, while maintaining a higher adsorption density and residual activity levels. The method demonstrated in this study will be applicable to the isolation of a variety of material-binding peptides against the surfaces of unique materials.     

General method for site-specific protein immobilization by Staudinger ligation

Protein microarrays are playing an increasingly important role in the discovery and characterization of protein-ligand interactions. The uniform orientation conferred by site-specific immobilization is a demonstrable advantage in using such microarrays. Here, we report on a general strategy for fabricating gold surfaces displaying a protein in a uniform orientation. An azido group was installed at the C-terminus of a model protein, bovine pancreatic ribonuclease, by using the method of expressed protein ligation and a synthetic bifunctional reagent. This azido protein was immobilized by Staudinger ligation to a phosphinothioester-displaying self-assembled monolayer on a gold surface. Immobilization proceeded rapidly and selectively via the azido group. The immobilized enzyme retained its catalytic activity and was able to bind to its natural ligand, the ribonuclease inhibitor protein. This strategy provides a general means to fabricate microarrays displaying proteins in a uniform orientation.     

A chemoselective method for site-specific immobilization of peptides via aminooxy group

Site-specific modification of peptides and proteins is an important area of basic research for preparation of well-defined biosensors and probes. The unique properties of aminooxy group present an opportunity for chemoselective site-specific immobilization of peptides to prepare well-defined biosensors. We have prepared FLAG peptide derivatives containing L-epsilon-aminooxylysine (L-epsilon-AOLys, 1a) and L-lysine units in their sequence at the C- and N-terminals via solid-phase synthesis. Site-specific modification of peptides through aminooxy group was demonstrated in the preparation of biosensors and selective conjugation in the preparation of biotinylated probes. Effect of the incorporation of L-epsilon-AOLys (1a) into the peptide sequence and its subsequent labeling on the FLAG epitopic character was measured using a surface plasmon resonance detector. It was found that incorporation of L-epsilon-AOLys (1a) into the FLAG peptide and site-specific immobilization through aminooxy group preserved the integrity of FLAG epitope.     

Metal-induced changes in the fluorescence properties of tyrosine and tryptophan site-specific mutants of oncomodulin.

Oncomodulin is a 108-residue, oncodevelopmental protein containing two calcium-binding sites identified as the CD- and EF-loops. The protein contains no tryptophan and only two tyrosine residues, one which is a calcium ligand in the CD-loop (Tyr-57) and one which lies in the flanking D-helix of this loop (Tyr-65). Site-specific mutagenesis was performed to yield five mutants, two with phenylalanine substituted for tyrosine in positions 57 and 65 and three with tryptophan substituted into position 57 in the CD-loop, position 65 in the D-helix, and position 96 in the EF-loop. The single Tyr-containing mutants demonstrated that position 57 was perturbed to a significantly greater extent than position 65 upon calcium binding. Although both tyrosine residues responded to decalcification, the fluorescence intensity changes were in opposite directions, with the more dominant Tyr-57 accounting for the majority of the intrinsic fluorescence observed in native oncomodulin. The substitution of tryptophan for each tyrosyl residue revealed that in both positions the tryptophan resided in polar, conformationally heterogeneous environments. The environment of Trp-57 was affected by Ca2+ binding to a much greater extent compared to that of Trp-65. Only 1 equiv of Ca2+ was required to produce greater than 70% of the Trp fluorescence changes in positions 57 and 65, indicating that Ca2+ binding to the higher affinity EF-loop had a pronounced effect on the protein structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioluminescence of outer membrane defective mutants of Photobacterium phosphoreum

Abstract Mutants of Photobacterium phosphoreum NCMB7 hypersensitive to several antimicrobiol agents were independently isolated and characterized. The hypersensitivity was probably due to changes in the outer membrane (OM) structure; the electrophoretic lipopolysaccharide profile of one mutant was altered. In addition, 2 mutants exhibited elevated cell surface hydrophobicity. With respect to bioluminescence, the mutants were dim or dark, and showed strongly reduced activities of the inducible enzymes luciferase and fatty acid reductase. Our results suggest that the outer membrane is involved in the regulation of bacterial bioluminescence.     

Characterization of recombinant human single chain urokinase-type plasminogen activator mutants produced by site-specific mutagenesis of lysine 158

The cDNA encoding full-length single chain urokinase-type plasminogen activator (scu-PA) was cloned and sequenced, and the recombinant scu-PA (rscu-PA) was expressed in Chinese hamster ovary cells. Two mutants, constructed by in vitro site-specific mutagenesis of Lys158 in rscu-PA to Gly158 (rscu-PA-Gly158) or to Glu158 (rscu-PA-Glu158), were also expressed in Chinese hamster ovary cells. Wild type and mutant rscu-PAs were purified to homogeneity by immunoadsorption on an insolubilized monoclonal antibody raised against natural scu-PA (nscu-PA), followed by gel filtration. The specific activity of the mutant scu-PAs on fibrin plates is very low (less than 1,000 IU/mg) compared to that of the wild type rscu-PA (44,000 IU/mg). The mutants, in contrast to the wild type rscu-PA, are not converted to amidolytically active two chain u-PA (tcu-PA) by plasmin and do not cause lysis of a 125I-fibrin-labeled plasma clot immersed in citrated plasma. However, in a purified system, both rscu-PA-Gly158 and rscu-PA-Glu158 activate plasminogen following Michaelis-Menten kinetics, with a much lower affinity (Km = 60-80 microM) but with a higher turnover rate constant (k2 = 0.01 s-1) as compared to the wild type rscu-PA (Km = 1.0 microM, k2 = 0.002 s-1). We conclude that conversion of scu-PA to tcu-PA is not a prerequisite for the activation of plasminogen. Substitution of Lys158 by Gly158 or Glu158 does, however, markedly decrease the stability of the Michaelis complex.     

Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails

Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in E. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycolbis (beta-aminoethyl) tetraacetic acid (EGTA) charged with Zn(2+) as precipitant, whereas protein A, carrying only one attached histidine tail, did not precipitate. However, all of the engineered proteins could be purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn(2+). The potential of using the same histidine tails for site-specific immobilization of proteins was also investigated. The enzymes were all catalytically active when immobilized on IMAC gels. For instance, immobilized lactate dehydrogenase, carrying tails composed of four histidine residues, displaced 83% of the soluble enzyme activity. (c) 1996 John Wiley & Sons, Inc.     

Thermostable mutants of the photoprotein aequorin obtained by in vitro evolution

Aequorin is a photoprotein that emits light upon binding calcium. Aequorin mutants showing increased intensity or slow decay of bioluminescence were isolated by in vitro evolution combining DNA shuffling and functional screening in bacteria. Luminescence decay mutants were isolated at the first round of screening and carried mutations located in EF-hand calcium binding sites or their vicinity. During in vitro evolution, the luminescence intensity of the population of mutants increased with the frequency of effective mutations whereas the frequency of other amino acid substitutions remained roughly stable. Luminescence intensity mutations neighbored the His-16 or His-169 coelenterazine binding residues or were located in the first EF-hand. None of the selected mutants exhibited an increase in photon yield when examined in a cell-free assay. However, we observed that two mutants, Q168R and L170I, exhibited an increase of the photoprotein lifetime at 37 degrees C that may underlie their high luminescence intensity in bacteria. Further analysis of Q168R and L170I mutations showed that they increased aequorin thermostability. Conversely, examination of luminescence decay mutants revealed that the F149S substitution decreased aequorin thermostability. Finally, screening of a library of random Gln-168 and Leu-170 mutants confirmed the involvement of both positions in thermostability and indicated that optimal thermostability was conferred by Q168R and L170I mutations selected through in vitro evolution. Our results suggest that Phe-149 and Gln-168 residues participate in stabilization of the coelenterazine peroxide and the triggering of photon emission by linking the third EF-hand to Trp-129 and His-169 coelenterazine binding residues.     

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.     

Intein-mediated site-specific conjugation of Quantum Dots to proteins in vivo

We describe an intein based method to site-specifically conjugate Quantum Dots (QDs) to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH) domain with the N-terminus half of a split intein (I_N). The C-terminus half (I_C) of the intein was conjugated to QDs in vitro. I_C-QD's and RNA encoding PH-I_N were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR)-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes.     

Purification and site-specific immobilization of genetically engineered glucose dehydrogenase on thiopropyl-sepharose

The gene encoding glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was inserted in a plasmid 1.0 kb downstream from a lac promoter, resulting in a 70-fold higher production of the enzyme when expressed in Escherichia coli. A glucose dehydrogenase mutant containing a cysteine residue at position 44 could also be expressed at the same high level. This single cysteine residue was used as an ‘affinity tag’ to simplify the purification procedure as well as for site-specific immobilization of glucose dehydrogenase on Thiopropyl-Sepharose. This enzyme was purified to homogeneity with a final recovery of 65% and a specific activity of 240 U/mg. The oriented immobilization resulted in increased thermal stability.     

FTIR study of secondary structure changes in Epidermal Growth Factor by gold nanoparticle conjugation

Conformation of protein is vital to its function, but may get affected when processing to manufacture products. It is therefore important to understand structural changes during each step of production. In this study, we investigate secondary structure changes in the targeting protein Epidermal Growth Factor (EGF) during synthesis of theranostic bifunctional nanoparticle, devised for Photodynamic therapy of breast cancer. We acquired FTIR spectra of EGF; unconjugated, post treatment with α-lipoic acid, attached to gold nanoparticle, and bound to the bifunctional nanoprobe. We observed decreasing disordered structures and turns, and increasing loops, as the synthesis process progressed. There was an overall increase in β-sheets in final product compared to pure EGF, but this increase was not linear and fluctuated. Previous crystal structure studies on EGF-EGFR complex have shown loops and β-sheets to be important in the binding interaction. Since our study found increase in these structures in the final product, no adverse effect on binding function of EGF was expected. This was confirmed by functional assays. Such studies may help modify synthesis procedures, and thus secondary structures of proteins, enabling increased functionality and optimum results.     

Isolation and properties of various molecular forms of aequorin.

The photoprotein aequorin emits light by an intramolecular reaction when a trace of Ca2+ is added. The samples of aequorin that were purified by the conventional methods of column chromatography were separated by high-performance liquid chromatography into eight molecular forms (isoaequorins), which were designated aequorins A-H. Aequorins A, C and F were obtained in crystalline states. A wide range of properties were studied with aequorins A-F, which were essentially pure. These six isoaequorins showed relatively small differences in their spectroscopic properties, but their values of A0.1%/1 cm, 280 were found to be close to 3.0, about 10% more than the previously reported value of 2.70-2.71 that was obtained with the samples of conventionally purified aequorin. The Mr values ranged from 20,100 (aequorin F) to 22,800 (aequorin A), the luminescence activities ranged from 4.35 X 10(15) photons/mg (aequorin A) to 5.16 X 10(15) photons/mg (aequorin F), and the first-order reaction rate constants of luminescence ranged from 0.95 s-1 (aequorin A) to 1.33 s-1 (aequorin F). As regards sensitivity to Ca2+, aequorin D was the most sensitive, having a sensitivity about 0.4-0.5 pCa unit above that of the least sensitive kind (aequorin A).     

Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation

Ligation-independent cloning (LIC) allows for cloning of DNA constructs independent of insert restriction sites and ligases. However, any required mutations are typically introduced by additional, time-consuming steps. We present a rapid, inexpensive method for mutagenesis in the 5′ LIC site of expression constructs and report on the construction of expression vectors with N-terminal serine, cysteine, threonine, or tyrosine residues after tobacco etch virus (TEV) protease cleavage. In a practical application, the N-terminal serine was oxidized to an aldehyde, subsequently reacted with an amino-oxy functionalized polyethylene glycol (PEG) ligand under aniline catalysis to provide a protein selectively modified at the N-terminus.