Studies on a mammalian cell protein (P8) with affinity for DNA in vitro

A protein (P8) found in cultured mammalian cells has been highly purified by DNA—cellulose chromatography alone. The protein is quite abundant, especially in human diploid fibroblasts, is readily extractable at low-salt concentration, and has an isoelectric pH close to neutrality. Its synthesis is not coupled to that of DNA, but it accounts for a greater proportion of the total soluble protein synthesis in growing than in resting cells. Among the proteins identified after DNA—cellulose chromatography, only P8 has affinity for single-stranded DNA but not for double-stranded DNA. It appears to have properties different from those of other DNA-binding proteins that have been described.     

A new affinity ligand for the isolation of a single 'feruloyl esterase' (FAE-III) from Aspergillus niger

8-Aminooctyl 5'-S-coniferyl-5'-deoxy-thio-alpha-L-arabinofuranoside has been synthesised and shown to be a selective affinity ligand for the feruloyl esterase III of Aspergillus niger. The hydrolyses of methyl 5-O-coumaroyl, feruloyl, or sinapoyl alpha-L-arabinofuranosides by this enzyme proceed at comparable rates.     

Synthetic biology for mammalian cell technology and materials sciences

The synthetic reconstruction of natural gene networks and the de novo design of artificial genetic circuits provide new insights into the cell's regulatory mechanisms and will open new opportunities for drug discovery and intelligent therapeutic schemes. We will present how modular synthetic biology tools like repressors, promoters and enzymes can be assembled into complex systems in order to discover small molecules to shut off antibiotic resistance in tubercle bacteria and to design self-sufficient therapeutic networks. The transfer of these synthetic biological modules to the materials science field enables the construction of novel drug-inducible biohybrid materials for biomedical applications.     

Affinity cross-flow filtration: purification of IgG with a novel protein a affinity matrix prepared from two-dimensional protein crystals

In this article, we describe the use of 1- to 2-mum sized affinity microparticles for the isolation and purification of IgG from artificial IgG-human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross-flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111-69, in which the peptidoglycan-containing layer was completely covered with a hexagonally ordered S-layer lattice. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S-layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S-layer protein release under cross-flow conditions between pH 2 to 12. The IgG-binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes. (c) 1994 John Wiley & Sons, Inc.     

Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures

Various DNA methyl transferase inhibitors (iDNMTs) and histone deacetylase inhibitors (iHDACs) were screened for their ability to enhance transient gene expression (TGE) in Human Embryonic Kidney 293-EBNA (HEK293E) cells. The effects in HEK293E cells were compared to those in Chinese Hamster Ovary DG44 (CHO-DG44) cells. The iDNMTs and iHDACs were chosen based on their different cellular activities and mechanisms of action. For each inhibitor tested, the optimum concentration was determined for both cell lines, and these conditions were used to evaluate the effect of each compound using a recombinant monoclonal antibody as a reporter protein. All the iHDACs increased transient antibody yield at least 4-fold in HEK293E and at least 1.5-fold in CHO-DG44. By comparison, the iDNMTs increased antibody yields by a maximum of approximately 2-fold. Pairwise combinations of iDNMTs and iHDACs had a linearly additive effect on TGE in CHO-DG44 but not in HEK293E. With valproic acid (VPA), volumetric and specific productivities of 200 mg/L and 20 pg/cell/day, respectively, were achieved in HEK293E cells with a 10-day process. As VPA is both FDA-approved and 5-fold less expensive than sodium butyrate (NaBut), we recommend it as a cost-effective alternative to this widely used enhancer of recombinant protein production from mammalian cells.     

CFD-aided design of a dynamic filter for mammalian cell separation

In the present work, a rotating disk filter was designed for mammalian cell separation with the aim of avoiding both cell damage and membrane fouling. Different geometric and operational variables of the rotating disk filter were studied using computational fluid dynamics (CFD) by varying rotor radius, rotor angle, membrane-rotor distance, and angular velocity. The combinations of these variables followed a statistical design, so that an analysis of the CFD results provided correlations describing the average shear stress on the membrane surface and the maximum shear stress in the whole module as a function of the variables studied. Based on these correlations, and on the shear resistance levels of Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cell lines, which were investigated using a cone-and-plate viscosimeter, it was possible to determine the geometry and angular velocity that would minimize both cell damage and membrane fouling. After construction, the filter was tested in filtration experiments at increasing permeate fluxes. Cell viability remained >90% for the duration of the experiments (2.5 h), and no indication of fouling was observed. It was shown that the designed dynamic filter is able to effectively avoid both cell damage and membrane fouling, and thus can be used for mammalian cell harvesting and perfusion.     

Aflatoxin B(1): Cytotoxic mode of action evaluated by mammalian cell cultures

Aflaxton B(1) rapidly inhibits RNA synthesis in rat liver cells, slices or liver in vivo. Established human cells lines (kidney T-cells, HeLa S(3), Chang liver) and mouse fibroblast 3t3 are more slowly affected. Prolonged exposure of synchronized cell cultures to the agent show that cells are retarded in their passage through the S-phase and exhibit a decreased rate of DNA synthesis. Consequent to this, mitosis is also inhibited. Liver cells appear to convert aflatoxin B(1) to a more potent cytotoxin which can then affect normally non-susceptible cells. This may explain the susceptibility of liver to tumorogenesis by this carcinogen.     

Method for isolation of intact titin (connectin) molecules from mammalian cardiac muscle

Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655–658). Small pieces of cardiac muscle were incubated for 2–3 weeks at 4°C in Ca²⁺-depleting solution before their homogenization to decrease activity of Ca²⁺-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119–123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca²⁺-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ～5–20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ～30–50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca²⁺-dependent proteases.     

Single-step Strep-tag purification for the isolation and identification of protein complexes from mammalian cells

Identification of protein complexes is the key to understanding cellular functions. In this study, we present a novel method for the identification of multiprotein complexes from mammalian cells. By using the Strep-tag affinity chromatography method, enabling fast and simple one-step purification, coupled with competitive elution under physiological conditions, we successfully purified a PP2A holoenzyme protein complex from a cultured mammalian cancer cell line. We identified, by mass spectrometry, both known and novel interacting proteins for PP2A, and demonstrate that the purified PP2A complex is functional. The benefits and potential applications of the Strep-tag method for protein complex purification are discussed.     

Constitutive secretion of an endogenous insulin-like peptide binding protein with high affinity for insulin in Spodoptera frugiperda (Sf9) cell cultures

Serum-free medium from batch cultures of Sf9 insect cells was examined for the occurrence of proteins related to the insulin-like growth factor family. We found that the Sf9 cell line constitutively produced and secreted a soluble protein with a MW of 27 kDa that exerted specific binding to human insulin-like growth factor-I (IGF-I) and -II. Moreover, the secreted protein bound human insulin and human proinsulin with higher affinity than IGF-I and -II. The order of affinity to the insulin peptides, determined by competitive inhibition of ligand binding, was: insulin > proinsulin > IGF-I > IGF-II. The dissociation constant (k(d)) for IGF-II was 28.5 +/- 1.7 nM and for insulin 7.2 +/- 1.3 nM, as determined by Scatchard plot analysis. The results suggest that the Sf9 cells produce an insulin binding protein similar to the human insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1).     

High rate shear strain of three-dimensional neural cell cultures: a new in vitro traumatic brain injury model

The fidelity of cell culture simulations of traumatic brain injury (TBI) that yield tolerance and mechanistic information relies on both the cellular models and mechanical insult parameters. We have designed and characterized an electro-mechanical cell shearing device in order to produce a controlled high strain rate injury (up to 0.50 strain, 30 s(-1) strain rate) that deforms three-dimensional (3-D) neural cultures (neurons or astrocytes in an extracellular matrix scaffold). Theoretical analysis revealed that these parameters generate a heterogeneous 3-D strain field throughout the cultures that is dependent on initial cell orientation within the matrix, resulting in various combinations of normal and shear strain. The ability to create a linear shear strain field over a range of input parameters was verified by tracking fluorescent microbeads in an acellular matrix during maximal displacement for a range of strains and strain rates. In addition, cell death was demonstrated in rat cortical astrocytes and neurons in response to high rate, high magnitude shear strain. Furthermore, cell response within the 3-D neuronal cultures depended on orientation, with higher predicted shear strain correlating with an increased loss of neurites, indicating that culture configuration may be an important factor in the mechanical, and hence cellular, response to traumatic insults. Collectively, these results suggest that differential responses exist within a 3-D culture subjected to mechanical insult, perhaps mimicking the in vivo environment, and that this new model can be used to investigate the complex cellular mechanisms associated with TBI.     

Assessment of a disc stack centrifuge for use in mammalian cell separation

A prototype disc stack centrifuge was tested for the separation of mammalian cell cultures from 80- and 2000-L fermentations. The clarification capacity for mammalian cells was excellent, but some smaller particles remained in the supernatant and reduced its usefulness for downstream processing. In order to identify the source of such particle formation, several parameters were assessed and minimum particle size for separation was calculated. An analysis of particle distribution was performed. Temperature and pressure effects inside the centrifuge bowl were measured. Some modifications of mechanical engineering can be suggested for the improvement of the use of standard disc stack centrifuges for mammalian cells. (c) 1995 John Wiley & Sons, Inc.     

Structural studies on a low oxygen affinity hemoglobin from mammalian species: Sheep (Ovis aries)

Hemoglobin (Hb) is in equilibrium between low affinity Tense (T) and high affinity Relaxed (R) states associated with its unliganded and liganded forms, respectively. Mammalian species can be classified into two groups on the basis of whether they express ‘high’ and ‘low’ oxygen affinity Hbs. Although Hbs from former group have been studied extensively, a limited number of structural studies have been performed for the low oxygen affinity Hbs. Here, the crystal structure of low oxygen affinity sheep methemoglobin (metHb) has been determined to 2.7 Å resolution. Even though sheep metHb adopts classical R state like quaternary structure, it shows localized quaternary and tertiary structural differences compared with other liganded Hb. The critical group of residues in the “joint region”, shown as a major source of quaternary constraint on deoxyHb, formed unique interactions in the α1β2/α2β1 interfaces of sheep metHb structure. In addition, the constrained β subunits heme environment and the contraction of N-termini and A-helices of β subunits towards the molecular dyad are observed for sheep metHb structure. These observations provide the structural basis for a low oxygen affinity and blunt response to allosteric effector of sheep Hb.     

3-Hydroxybenzoate:coenzyme A ligase from cell cultures of Centaurium erythraea: isolation and characterization

In xanthone biosynthesis, 3-hydroxybenzoate:coenzyme A ligase (3HBL) supplies the starter substrate for the formation of an intermediate benzophenone. 3HBL from cell cultures of the medicinal plant Centaurium erythraea was purified to apparent homogeneity using a seven-step-procedure. The enzyme was an AMP-forming CoA ligase with a Km = 14.7 microM for 3-hydroxybenzoic acid, 8.5 microM for coenzyme A and 229 microM for ATP. The pH and temperature optima were 7.5 and 35 degrees C, respectively. In SDS-PAGE, two polypeptides of Mr 41,500 and 40,500 were detected. Both proteins were structurally related to each other as shown by tryptic digestion. Their N-termini were blocked. The difference in their apparent molecular masses could not be attributed to glycosylation. 3HBL had a native Mr of approx. 50,000 and is thus active as a monomer.