Expression and characterization of the human c-myc DNA-binding protein.

In an effort to study in detail the nature of the protein product of the human protooncogene c-myc, we have expressed the gene at high levels in Escherichia coli. The c-myc coding region was taken from a full-length cDNA clone and inserted into a vector designed to express foreign gene products efficiently in E. coli. Pulse-labeling experiments indicated that the rate of expression of c-myc in this thermoinducible expression system is very efficient. The product was relatively stable and accumulated to approximately 10% of total cellular protein. A purification protocol was devised which allowed the c-myc protein to be readily purified in quantities sufficient for detailed biochemical and physical analyses. A high-titer polyclonal antiserum was raised against the pure protein and shown to immunoprecipitate the p110gag-myc fusion protein of MC-29-infected quail cells. This antiserum also selectively detects a protein with an apparent molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis from a Burkitt lymphoma cell line. We conclude that this 64-kilodalton protein is the human c-myc gene product since the E. coli-made protein exhibits an equivalent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even though its calculated molecular weight is 49,000. Furthermore, we demonstrate that the bacterially made human c-myc protein is a DNA-binding protein and that it exhibits a high nonspecific affinity for double-stranded DNA.

Metal-binding characteristics of the parotid salivary protein gustin.

Metal binding characteristics of the parotid salivary protein gustin have been examined. When purified to apparent homogeneity, gustin contains 1 gatom Zn/mol which is tightly bound (Kd at pH 7.2, 4.5--10(-11) M). This tightly bound zinc can be removed with strong chelators such as diethyldithiocarbamic acid and 1,10-o-phenanthroline at pH 4.5, but not with EDTA or Chelex 100. Removal of the metal ion causes no appreciable conformational change in the protein. The apoprotein can be reconstituted by dialysis against Zn2+-containing buffer, a process favored by pH greater than 6.0. Only cobalt is able to bind to the apoprotein at this strong binding site. Cobalt binding is appreciably weaker than that of zinc (Kd at pH 7.2, 1.3--10(-7) M) and is maximal at pH 7.0. The weaker binding of cobalt is also illustrated by the loss of 37% of bound cobalt after 96 h of dialysis at pH 7.2, conditions under which the zinc content of gustin does not change. A second gatom Zn/mol may be loosely bound to gustin, but is easily removed by dialysis against metal ion-free buffer. Other metal ions such as copper, nickel, iron or manganese, but not cadmium or mercury, bind loosely to this second zinc site and are removed with ease. Zinc appears to be involved in the formation of the complex between gustin and glycoproteins which are present in human parotid saliva in vivo.

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides.

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.

Production of monoclonal antibodies in COS and CHO cells.

Recent advances in the generation of genetically engineered monoclonal antibodies have enhanced the importance of COS cells as expression systems for rapidly producing sufficient quantities of these proteins for preliminary biochemical and biophysical analysis. In order to meet the demand for clinical supplies, a gradual increase has occurred in the usage of dihydrofolate reductase negative (DHFR-) Chinese hamster ovary (CHO) cells for large-scale antibody production. Using a variety of mammalian expression vectors and selection/amplification protocols, CHO cell lines capable of producing monoclonal antibodies at levels exceeding 1 gl-1 can now be obtained in an almost routine fashion. For the applications of monoclonal antibodies to expand into additional therapeutic areas, however, a 5-10-fold increase over current highest expression levels may still need to be achieved.

High-level production of biologically active human cytosolic phospholipase A2 in baculovirus-infected insect cells.

Infection of Spodoptera frugiperda insect cells with a recombinant baculovirus expressing human cytosolic phospholipase A2 (cPLA2) resulted in the production of biologically active protein. The level of recombinant human cPLA2 production in infected insect cells was at least 50-fold higher than that observed in human monoblast U937 cells.

Plasmid pFCE4: a new system of Escherichia coli expression-modification vectors.

Two versatile expression-modification vectors were obtained by inserting the origin of replication (ori) of phage f1 into the expression vector pOTS. The resulting plasmids produce large amounts of coding or noncoding ssDNA (depending on ori orientation in pFCE4+ and pFCE4-) and excrete it into the medium as virus-like particles following infection with phage f1. These features make them suitable for dideoxy chain termination sequencing, oligonucleotide directed mutagenesis and gene expression without further manipulations. The human IFN alpha-2 gene, lacking the codon for the first amino acid, cysteine, was efficiently expressed by these vectors.

Quantitative Western blot assay for measurement of the murine acute phase reactant, serum amyloid P component.

Quantitation of the murine acute-phase reactant, serum amyloid P component (SAP), by Western blot is described. The assay is sensitive, reliable and inexpensive. Electrophoresis in standard SDS-polyacrylamide gels (SDS-PAGE) effectively separates SAP from other serum proteins. Electrophoretic blotting of SAP from the SDS-PAGE onto nitrocellulose (NC) paper is followed by a bovine serum albumin 'blocking' wash and exposure to anti-SAP antibody. Subsequent incubation with radioiodinated protein A was followed by autoradiography, and SAP bands were cut from the NC paper and counted in a gamma counter. The utility of this method for quantitation of SAP in biological fluids was verified using sera from normal mice and mice undergoing an acute inflammatory response. The results confirm the elevation of SAP associated with acute inflammation. The sensitivity of this technique coupled with the minute volumes of biological sample required renders it of potential utility for SAP quantitation in a variety of inflammatory disease states.

Long-term effects of radiotherapy on taste and salivary function in man.

The long-term effects of radiotherapy on taste and salivary function were studied in 13 patients treated by radiation 1--7 years previously for tumors of the head and neck. Taste function was quantitatively evaluated using a standard forced choice, three-stimulus-drop technique for the determination of detection and recognition thresholds and a forced-scaling technique for the determination of taste intensity responsiveness. Parotid salivary function was quantitatively evaluated by determination of flow rate and protein secretion rate. Nine of the 13 patients studied (69%) had measurable taste loss; every patient who had radiotherapy including the parotid glands had measurable salivary dysfunction. Our results demonstrate that curative courses of radiotherapy for tumours of the head and neck may result in long-term changes in taste and salivary function. From the present study, the maximum tolerance doses resulting in a 50% complication rate 5 years after treatment (TD 50/5) are estimated to be 40--65 Gy for xerostomia and 50--65 Gy for taste loss. Therefore, in a standard treatment regimen for tumors of the head and neck, with curative intent, gustatory and salivary gland tissues frequently sustain maximum tolerance injury.

The genetic analysis of the HIV envelope binding domain on CD4.

Through mutagenesis, we identified a single high-affinity binding site for gp120 on the human CD4 protein. This site is localized in the V1 domain within residues 41 to 55. The collection of mutants was also used to define the epitopes for 55 anti-CD4 monoclonal antibodies. The locations of these epitopes are consistent with a V kappa-like structure for the V1 domain. In the context of this structure, the gp120 binding site encompasses the small CDR2 loop. Through deletion mutagenesis at the termini of the V1 domain, we further defined the minimal region required to retain high-affinity binding to gp120. Short deletions at both termini disrupt binding to gp120 and recognition by conformation-sensitive anti-CD4 monoclonal antibodies. We conclude that amino acids at both the amino and carboxy termini are critical to the conformation of the V1 domain and, in particular, to the integrity of the gp120 binding site.

Effector-assisted refolding of recombinant tissue-plasminogen activator produced in Escherichia coli.

Recombinant tissue-plasminogen activator (r-tPA), expressed in Escherichia coli cells in an aggregated form, was solubilized with a strong chaotrope in the absence of any reducing agent. The solubilized molecule was reactivated by a procedure that was developed to mimic the physiological conditions optimal for the functional folding and activity of the native protein. The use of partially purified fibrinogen, as a source of fibrin (the effector), is shown to facilitate the reactivation process and increase its yield by at least a factor of two. The yield of the process is also shown to be particularly dependent on the recombinant protein concentration. At a concentration level of 3-3.7 mg r-tPA/L in the reactivation mixture, up to a 90% yield of activity was obtained. Purification of the activated form of r-tPA was achieved with a two-step column-chromatography scheme. This included a gel filtration step on a Sephadex G-50 column followed by an affinity chromatography step on a lysine-sepharose column. The product was composed of roughly equal amounts of one-chain and two-chain t-PA. The feasibility of using a two water-soluble polymeric phase system, with a centrifugal partition chromatography (CPC), in scaling up the reactivation process or the purification step was also evaluated.

The proline-, glycine-, glutamic acid-rich pink-violet staining proteins in human parotid saliva are phosphoproteins.

The major proteins in human parotid saliva, isolated in Fractions II-V following chromatography on Sephacryl S-200, DEAE-Sephadex A-50, or CM cellulose, contain 6 moles of phosphate per mole of protein, the phosphate probably bound to the protein via an ester linkage. This phosphate represents greater than 90% of the protein-bound phosphate in human parotid saliva. Neither purified gustin nor amylase, the two other major proteins in human parotid saliva, contain phosphate.

Biosynthesis and cellular distribution of the two superoxide dismutases of Dactylium dendroides.

The synthesis and subcellular localization of the two superoxide dismutases of Dactylium dendroides were studied in relation to changes in copper and manganese availability. Cultures grew normally at all medium copper concentrations used (10 nM to 1 mM). In the presence of high (10 muM) copper, manganese was poorly absorbed in comparison to the other metals in the medium. However, cells grown at 10 nM copper exhibited a 3.5-fold increase in manganese content, while the concentration of the other metals remained constant. Cultures grown at 10 nM copper or more had 80% Cu/Zn enzyme and 20% mangani enzyme; the former was entirely in the cytosol, and the latter was mitochondrial. Removal of copper from the medium resulted in decreased Cu/Zn superoxide dismutase synthesis with a concomitant increase in the mangani enzyme such that total cellular superoxide dismutase activity remained constant. The mangani enzyme in excess of the 20% was present in the non-mitochondrial fraction. The mitochondria, therefore, show no variability with respect to superoxide dismutase content, whereas the soluble fraction varies from 100 to 13% Cu/Zn superoxide dismutase. Copper-starved cells that were synthesizing predominantly mangani superoxide dismutase could be switched over to mostly Cu/Zn superoxide dismutase synthesis by supplementing the medium with copper during growth. Immunoprecipitation experiments suggest that the decrease in Cu/Zn activity at low copper concentration is a result of decreased synthesis of that protein rather than the production of an inactive apoprotein.