Stable self-assembly of a protein engineering scaffold on gold surfaces

The outer membrane protein OmpF from Escherichia coli is a member of a large family of beta-barrel membrane proteins. Some, like OmpF, are pore-forming proteins whilse others are active transporters or enzymes. We have previously shown that the receptor-binding domain (R-domain) of the toxin colicin N binds with high affinity to OmpF reconstituted into tethered lipid bilayers on gold electrodes. The binding can be measured by surface plasmon resonance (SPR) and ion channel blockage (impedance spectroscopy, IS). In this paper we report the use of a mutant OmpF-E183C in which a single cysteine had been introduced on a short periplasmic turn. OmpF-E183C binds directly to gold surfaces and creates high-density protein layers by self-assembly from detergent solution. When the gold surface is pretreated with beta-mercaptoethanol and thiolipids are added after the protein immobilisation step, the protein is shown, by Fourier transform infrared spectroscopy (FTIR), to retain its beta-rich structure. Furthermore, we could also measure R-domain binding by SPR and IS, confirming the functional reconstitution of a self-assembled membrane protein monolayer at the gold surface. Because these beta-barrel proteins are recognized protein engineering scaffolds, the method provides a generic method for the simple self-assembly of protein interfaces from aqueous solution.     

The M. tuberculosis antigen 85 complex and mycolyltransferase activity

AIMS: The antigen 85 complex (Ag85) from Mycobacterium tuberculosis consists of three abundantly secreted proteins (FbpA, FbpB and FbpC2) which play a key role in the pathogenesis of tuberculosis and also exhibit cell wall mycolyltransferase activity. A related protein with similarity to the Ag85 complex was recently annotated in the M. tuberculosis genome as FbpC1. An investigation was carried out to determine whether FbpC1 may also possess mycolyltransferase activity, a characteristic feature of the Ag85 complex. METHODS AND RESULTS: Heterologous expression of FbpA, FbpC1 and FbpC2 was performed in Escherichia coli. Recombinant proteins were purified under non-denaturating conditions and used in an in vitro mycolyltransferase assay. CONCLUSIONS: In contrast to FbpA and FbpC2, recombinant FbpC1 did not possess in vitro mycolyltransferase activity and was not recognized by two monoclonal antibodies to the native Ag85. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycolyltransferase activity is restricted to FbpA, FbpbB and FbpC2 only; the actual function of FbpC1 remains to be established.     

Expression, Purification, and Bioassay of Human Stanniocalcin from Baculovirus-Infected Insect Cells and Recombinant CHO Cells

Stanniocalcin is a calcium- and phosphate-regulating glycoprotein hormone that was first described in fish where it functions in preventing hypercalcemia. Human cDNA clones encoding the homolog of stanniocalcin have been recently isolated. In this study, the full-length cDNA coding for human stanniocalcin (hSTC) was cloned into both baculovirus and CHO expression vectors. Recombinant hSTC was then produced efficiently from both baculovirus-infected insect cells and CHO cells in large-scale bioreactors. Purification protocols were developed and used to purify recombinant hSTC from both sources in four chromatography steps. The hSTCs from both expression systems were secreted as glycosylated proteins and as disulfide-linked homodimers. The results from glycosylation studies indicated that stanniocalcin from both sources contained N-linked oligosaccharides but no O-linked sugars. In anin vivobioassay based on the inhibition of gill calcium transport in fishes, the baculovirus and CHO-expressed protein showed biological activity which is dose dependent. The inhibitory effects of hSTC produced from both systems were essentially equipotent in fishes, despite the differences in glycosylation. Consequently, the precise role of the carbohydrate moiety in recombinant hSTC remains to be determined.     

Electron-energy-loss spectroscopic imaging of calcium and nitrogen in the cell walls of apple fruits

Changes in texture are an integral part of ripening in most fleshy fruits and these changes are thought to be determined, primarily, by alterations in cell wall structure. Electron energy loss spectroscopy (EELS) imaging was used to obtain quantitative information on the levels of calcium and nitrogen in the cell walls of apple (Malus domestica Borkh. cv. Cox's Orange Pippin) fruits. Samples of fruit cortex were prepared for EELS by high-pressure freezing and molecular distillation drying to minimize loss and redistribution of soluble cell wall components such as calcium. The EELS imaging successfully resolved calcium and nitrogen levels in the middle lamella and primary cell wall. When the elemental compositions of the cell walls of Cox's apples from two sites in the UK were compared at harvest or after 6 months storage, the orchard which always produced consistently firmer fruit had significantly lower levels of cell wall calcium and higher levels of cell wall nitrogen. This result was unexpected since firm texture in apples and other fruits has been commonly associated with elevated levels of fruit calcium. The nitrogen-rich material in the sections used for EELS was insoluble in acidified methanol, indicating that it represented a high-molecular-weight component in the cell wall. Furthermore, total tissue hydroxyproline levels were greatest in material with elevated cell wall nitrogen, suggesting enhanced levels of wall structural proteins in the tissue. These data indicate a correlation between increased amounts of cell wall nitrogen and firm fruit texture. The possible role of cell wall proteins in determining the textural properties of fruit tissue is discussed. Received: 19 November 1998 / Accepted: 28 January 1999     

TCR vaccines against a murine T cell lymphoma: a primary role for antibodies of the IgG2c class in tumor protection

Tumor-associated proteins can act as effective immunotherapeutic targets. Immunization with tumor TCR protein conjugated to the immunogenic protein keyhole limpet hemocyanin (KLH) protects mice from tumor challenge with the murine T cell lymphoma C6VL. The immune mechanisms responsible for this tumor protection are of interest for designing more effective vaccine strategies. Previous studies using depletion experiments had suggested a CD8-mediated component of protection induced by TCR-KLH vaccines. In this study we used CD8alpha knockout, micro MT, and FcgammaR knockout mice to investigate the relative roles of CD8+ T cells and Ab in protective immunity induced by TCR-KLH immunization. We found that CD8+ T cells are not required for tumor protection, although they may contribute to protection. Vaccine-induced Abs are sufficient to mediate protection against this murine T cell lymphoma through an FcR-dependent mechanism. This was confirmed with Ab transfers, which protect challenged mice. Additionally, recombinase-activating gene 1(-/-) splenocytes can mediate Ab-dependent cellular cytotoxicity against this tumor in the presence of bound anti-TCR Abs. IFN-gamma knockout mice demonstrated a requirement for IFN-gamma, probably via generation of IgG2c Abs, in vaccine-induced tumor protection. IFN-gamma knockout mice were not protected by immunization and had a severe impairment in IgG2c Ab production in response to immunization. Although mock-depleted anti-TCR Abs could transfer tumor protection, IgG2c-deficient anti-TCR Abs were unable to transfer tumor protection to wild-type mice. These results suggest that TCR-KLH vaccine-induced tumor protection in the C6VL system is primarily attributable to the induction of IgG2c Abs and humoral immunity.     

A finite element model for analyzing shear wave propagation observed in magnetic resonance elastography

Magnetic resonance elastography (MRE) is a novel non-invasive approach to determine material stiffness by using a conventional magnetic resonance imaging (MRI) system incorporated with an oscillating motion-sensitizing gradient to detect nodal displacements produced by a shear excitation wave. The effects of material properties, excitation frequency, boundary conditions, and applied tension on shear wavelength measurement must be examined before MRE can become a useful diagnostic tool. We propose finite element (FE) modeling as a robust method to systematically study the effects of these parameters. An axisymmetric FE model was generated with ABAQUS to simulate agarose gel phantoms. The effects of material stiffness, density, and excitation frequency on propagating shear wavelength were examined individually. The effect of the boundary conditions on shear wavelength was also demonstrated. Results of shear wavelength from MRE measurement were compared with the results of FE model, which showed good agreement between the methods.     

Identification of the testing parameters in high frequency dynamic shear measurement on agarose gels

Dynamic mechanical analysis (DMA) on agarose gels can be used to validate magnetic resonance elastography (MRE) measurements as well as to provide better understanding for the biological responses of cells to the dynamic loadings in cell culture studies. Various parameters potentially affecting the repeatability and accuracy of the DMA shear modulus measurements were investigated systematically in the present study, including sample thickness, shear strain, testing frequency, and compressive clamping strain. The study showed that the thickness of the agarose gel sample must be sufficiently small (1 mm) to prevent the erroneous fluctuation in the measured modulus. The appropriate levels of shear strain (< or = 0.5%) and compressive clamping strain (5-10%) must be applied to overcome the slippage at the gel-clamp interface without causing significant boundary and stress non-uniformity or micro-cracks in the agarose gel sample.