Dissolved oxygen concentration affects the accumulation of HIV-1 recombinant proteins in Escherichia coli

A central problem in aerobic growth of any culture is the maintenance of dissolved oxygen concentration (DOC) above growth-limiting levels especially in high-cell density fermentations that are usually of the fed-batch type. Fermentor studies have been conducted to determine the influence of DOC on the production of heterologous proteins in Escherichia coli. The results demonstrated that there is a significant degree of product-to-product variation in the response of heterologous protein accumulation to DOC. For translational fusions of the human immunodeficiency virus-1 (HIV-1) proteins p24Gag and Env41, the imposition of a dissolved oxygen (DO) limitation resulted in 100 and 15% increases in the respective product yields. On the other hand, the imposition of a DO limitation had no effect on the production of a similar translational fusion of the HIV-1 protein p55Gag, and a large negative effect on the production of an influenza protein (C13). The stimulatory effects of DOC on p24Gag production were investigated further. The results of my studies suggested that the stimulatory effect observed at reduced agitation rates on p24Gag accumulation was owing to an oxygen effect and not a shear effect. Furthermore, the results of my investigations indicated that the effect a DOC had on the production of p24Gag was strongly influenced by the cell density at which the culture was induced.     

Absolute quantitation of host cell proteins in recombinant human monoclonal antibodies with an automated CZE‐ESI‐MS/MS system

We report the first use of CZE for absolute characterization of host cell proteins (HCPs) in recombinant human monoclonal antibodies. An electrokinetically pumped nanoelectrospray interface was used to couple CZE with a tandem mass spectrometer. Three isotopic‐labeled peptides (LSFDKDAMVAR, VDIVENQAMDTR, and LVSDEMVVELIEK) were synthesized by direct incorporation of an isotope‐labeled lysine or arginine. The heavy‐labeled peptides were spiked in the HCP digests at known concentrations. After CZE‐ESI‐MS/MS analysis, the peaks of native and isotopic‐labeled peptides were extracted with mass tolerance ≤ 5 ppm from the electropherograms, and the ratios of peak area between native and isotopic‐labeled peptides pairs were calculated. Calibration curves (the ratios of peak area versus spiked peptide amount) with R² values of 0.999, 0.997, and 0.999 were obtained for the three HCP peptides, and the absolute amounts of the three proteins present were determined to be at the picomole level in a 20 μg sample of digested HCPs. The target proteins were present at the 7–30 ppt level in the purified HCP samples.     

Conjugation of proteins with chelating polymers via water-soluble carbodiimide andN-hydroxysulfosuccinimide

Water-soluble chelating polymers (CP) based on polylysine and diethylenetriaminepentaacetic acid (DTPA) have been prepared. The effect of the number of DTPA-groups in the polymer chain on the process of CP carbodiimide-mediated coupling to proteins has been studied. CP obtained were conjugated with proteins via carbodiimide andN-hydroxysulfosuccinimide (HSSI). The optimal conditions of CP activation were determined using model low-molecular-weight amine. It was shown that the addition of HSSI to an activation mixture increases the coupling efficiency of CP with immunoglobulins by 3‐4-fold compared with carbodiimide alone. Possible mechanisms of this phenomenon are discussed.     

Analysis of <Emphasis Type="Italic">O</Emphasis>-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis. In IgAN, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the galactose deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. We have previously demonstrated the first direct localization of multiple O-glycosylation sites on a single IgA1 myeloma protein by use of activated ion-electron capture dissociation (AI-ECD) Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry. Here, we report the analysis of IgA1 O-glycan heterogeneity by use of FT-ICR MS and liquid chromatography FT-ICR MS to obtain unbiased accurate mass profiles of IgA1 HR glycopeptides from three different IgA1 myeloma proteins. Additionally, we report the first AI-ECD fragmentation on an individual IgA1 O-glycopeptide from an IgA1 HR preparation that is reproducible for each IgA1 myeloma protein. These results suggest that future analysis of IgA1 HR from IgAN patients and normal healthy controls should be feasible.     

In vitro corrosion study of different TiO2 nanotube layers on titanium in solution with serum proteins

Titanium oxide nanotubes prepared by anodization have received considerable attention in the biomaterials domain. The objective of this study was to demonstrate the electrochemical behavior of different diameter TiO(2) nanotube layers on titanium in phosphate buffered saline (PBS) and Dulbecco's minimum essential medium+10% fetal calf serum (D-FCS) using open circuit potentials (OCP), electrical impedance spectroscopy (EIS), and a potentiodynamic polarization test. The results showed that the nanotubes had higher OCP, higher resistance of the inter barrier layer (R(b)), and lower I(pass) in the two test solutions compared to the smooth Ti, especially the 30 nm diameter nanotubes. The corrosion resistance of the nanotubes in D-FCS was higher than in PBS because of protein adsorption from the D-FCS solution as suggested by scanning electron microscope (SEM) images. In addition, protein aggregates of 30 nm diameter nanotubes caused the model of EIS spectra to transform from two-layer to three-layer. The corrosion behavior of the nanotubes for use as a dental implant material is discussed.     

Determination of total proteins in several tissues of rat: a comparative study among spectrophotometric methods

In the present paper, five spectrophotometric methods — biuret, Lowry modified by Hartree (Lowry/Hartree), p-chloranil, ultraviolet at 280 nm (UV-280 nm), and ultraviolet at 260/280 nm (UV-260/280 nm) — were used for protein determination in several rat tissues and the results were compared. The Lowry/Hartree method showed the highest sensitivity (0.9 μg/ml) and the biuret method the lowest sensitivity (55.7 μg/ml). The results were not statistically different for the following methods and tissues: Lowry/Hartree was compared to biuret for five tissues (adrenal, spleen, whole brain, liver and small intestine), to p-chloranil for two tissues (liver and epididymal fat pad), and to UV-260/280 nm for three tissues (whole brain, liver and pancreas). Since the Lowry/Hartree method needs a larger period of time for a whole assay to be carried out, we recommend for determination of total protein the following methods: UV-260/280 nm for whole brain, liver, and pancreas, p-chloranil for epididymal fat pad, and biuret for other tissues. We do not recommend the UV-280 nm method because there are many interfering substances in the tissues.     

Anab initio MO study on the direct and migratory reaction mechanism for the formation of HF in H + CIF system

Anab initio MO method has been used to calculate the potential energy surface for the formation of HF when H reacts with C1F. The various transition states possibly related to forming HF were optimized. An IRC calculation starting from the collinear F-centered transition state was performed and energies of a series of points with bending configurations were also calculated to determine the direct reaction path leading to HF product. Another IRC calculation starting from the H-centered transition state was run to determine the migratory path to forming HF. By doing so, the Polanyi’s assumption that it would involve the direct and a migratory mechanisms for the formation of HF when H reacts with CIF has been verified theoretically. Project supported by the National Natural Science Foundation of China.     

Time and temperature-based study for the production of high T C phase by sol-gel technique in Pb-bscco system

The high T_C superconducting phase Bi₂Sr₂Ca₂Cu₃Ox (2223) in the Pb-BSCCO system has been produced by EDTA-gel processing using nitrate solutions. The precursor has heated in two stages, at 300 and 800°C each for 2 h, to avoid the burning of the important species involved in the final product. The effects of time (6 to 48 h) and temperature (845 and 855°C) on the formation of the 2223 phase have been studied by sintering the samples in air. Thermal analysis (TG/DTA), X-ray diffraction (XRD), scanning electron microscopy (SEM) and a vibrating sample magnetometer (VSM) have been employed to investigate the powder produced at different stages of decomposition, oxidation and formation of sintered materials from the powders. The volume-fraction of the 2223 phase at 845°C increases with time, the maximum value of the 2223 phase was obtained at 120 h. It has been observed that the formation of the high T_C phase is remarkably enhanced at the temperature of the endothermic peak of the DTA curve. The best result has been obtained in the sample sintered for 24 h at the temperature 855°C (endothermic peak). This also indicated that at 855°C, the large volume-fraction of 2223 phase with T_C 113 K grew in short time and as the sintering time increased, it decomposed into the Bi₂Sr₂CaCu₂Ox (2212) phase and other phases.     

IR spectroscopic study of ion-molecular interactions in the methanesulfonic acid—N,N-dimethylformamide system

Complex formation in the methanesulfonic acid (MSA)-DMF system was studied by Multiple Attenuated Total Reflectance (MATR) IR spectroscopy at 30°C within the composition range from neat MSA up to neat DMF. Depending on the ratio of components, two types of complexes with a strong quasi-symmetrical H bond (1 and2) are formed. The uncharged complex1 is a quasi-ion pair with the (O…H…O) bridge. Complex2 is formed by a protonated DMF molecule and the (A…H…A)⁻ anion bound as an ion pair. It is established that complexes1 are solvated by DMF molecules in an excess of a base. Solvation or2 in an excess of an acid corresponds to a change from contact to contact-separated ion pairs. Continuous absorption spectra of charged and uncharged complexes1 and2 were obtained. The schemes of acid-base interactions in the MSA-DMF and HCl-DMF systems were compared. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2484–2489, December, 1998.     

Application of conduction calorimetry for study of the reactivity of C2S in the system C2S-C4A3¯S-C¯S-H

A conduction calorimeter was used to determine the effect of the ignition temperature on the hydration reactivity of C₂S in the system C₂S-C₄A₃¯S-C¯S relevant to sulphoaluminate belite cement. The results thus obtained showed that the kinetics and mechanism of hydration of the system C₂S-C₄A₃¯S-C¯S are influenced by the temperature of C₂S synthesis. The hydration reactivity of C₂S increases with increasing temperature of ignition.