Capture of human Fab fragments by expanded bed adsorption with a mixed mode adsorbent

A novel group of mixed mode adsorbents has been developed for purification of monoclonal and polyclonal antibodies from a broad range of raw materials such as hybridoma cell culture, ascites fluid, animal sera, milk, whey and egg yolk. The aim of this study was to determine whether such mixed mode adsorbents were also useful for the recovery of recombinant proteins from microbial feedstocks. This paper describes the performance of one of these adsorbents for expanded bed capture of a human Fab fragment from recombinant E. Coli cell extracts.It is concluded that the mixed mode adsorbent binds the Fab fragment efficiently from crude extracts without any requirement for preconditioning the extract by for example de-salting or dilution. The capacity of the mixed mode adsorbent is approx. 12 mg Fab/ml matrix.The novel mixed mode adsorbent can be useful during production of highly purified Fab fragments as the first step in a purification scheme. In this respect the mixed mode adsorbent is advantageous over alternative commercially available ion-exchange materials which require pre-conditioning of cell extract for Fab' capture. Together with the concentration and clarification effect a significant enrichment of the Fab fragment is obtained in one single high yield operation.     

Significance of kinetic degrees of freedom in operation of the actomyosin motor

The actomyosin motor as a principal functional component of cell motility is highly coordinated in regulating the participating molecular components. At the same time, it has to be flexible and plastic enough to accommodate itself to a wide variety of operational conditions. We prepared two different types of actomyosin systems. One is a natural intact actomyosin system with no artificial constraint on the kinetic degrees of freedom of the actin filaments, and the other is a regulated one with actin filaments supplemented by intra- and intermolecular crosslinking to suppress the kinetic degrees of freedom to a certain extent. Crosslinked actomyosin systems were found to remain almost insensitive to calcium regulation even when intact troponin-tropomyosin regulatory component was incorporated. Both the ATPase and the motile activities of the actin filaments sliding on myosin molecules were markedly lowered by the crosslinking. In contrast, once the crosslinking was cleaved, both properties returned to the normal as with intact actomyosin systems.     

Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F

Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.     

Effect of temperature and guanidine hydrochloride on ferrocytochrome<Emphasis Type="Italic"> c</Emphasis> at neutral pH

Thermally denatured horse heart ferrocytochrome c (ferrocyt c) has been characterized using absorption spectroscopy, differential scanning calorimetry (DSC) and viscometry at pH 7.0. DSC experiments have yielded the transition temperature of denaturant-free ferrocyt c unfolding as 100.6±0.3 °C, indicating an extremely high stability of the protein. The presence of guanidine hydrochloride (GdnHCl) facilitated estimation of the structural features of thermally unfolded ferrocyt c. The stability of the protein, expressed by G _D at 25 °C, is 59±5 kJ mol^–1 (DSC) and 65±6 kJ mol^–1 (absorption spectroscopy). An absorption spectrum of ferrocyt c demonstrates that the heme occurs in the high-spin state at extreme denaturing conditions (94 °C, 6.6 M GdnHCl). Absorption spectroscopy, using heme as a probe, shows that thermal denaturation of ferrocyt c occurs as a transition from a native low-spin (Met80/His18) to a high-spin disordered state with involvement of non-native, low-spin (bis-His) species.Abbreviations CD circular dichroism - cyt c cytochrome c - DSC differential scanning calorimetry - ferricyt c ferricytochrome c - ferrocyt c ferrocytochrome c - GdnHCl guanidine hydrochloride - NHE normal hydrogen electrode     

Microbial acetate oxidation in horizontal rotating tubular bioreactor

The aim of this work was to investigate the possibility of conducting a continuous aerobic bioprocess in a horizontal rotating tubular bioreactor (HRTB). Aerobic oxidation of acetate by the action of a mixed microbial culture was chosen as a model process. The microbial culture was not only grown in a suspension but also in the form of a biofilm on the interior surface of HRTB. Efficiency of the bioprocess was monitored by determination of the acetate concentration and chemical oxygen demand (COD). While acetate inlet concentration and feeding rate influenced efficiency of acetate oxidation, the bioreactor rotation speed did not influence the bioprocess dynamics significantly. Gradients of acetate concentration and pH along HRTB were more pronounced at lower feeding rates. Volumetric load of acetate was proved to be the most significant parameter. High volumetric loads (above 2 g acetate l⁻¹ h⁻¹) gave poor acetate oxidation efficiency (only 17 to 50%). When the volumetric load was in the range of 0.60–1.75 g acetate l⁻¹ h⁻¹, acetate oxidation efficiency was 50–75%. At lower volumetric loads (0.14–0.58 g acetate l⁻¹ h⁻¹), complete acetate consumption was achieved. On the basis of the obtained results, it can be concluded that HRTB is suitable for conducting aerobic continuous bioprocesses.     

Effects of dietary L-arginine supplementation on serum lipids and intestinal enzyme activities in diabetic rats

We investigated whether dietary supplementation with L-arginine, the endogenous precursor of nitric oxide, might affect serum lipid levels and activities of intestinal mucosa enzymes in animals, in which diabetes was induced by administration of streptozotocin. Control and diabetic rats were fed diets with or without 2% L-arginine supplementation for 4 weeks. Diabetic rats had significantly higher concentrations of serum triglycerides and LDL-cholesterol than control rats. These alterations were partially reduced by L-arginine supplementation. Experimental diabetes did not influence the lactase and leucine aminopeptidase activity in the intestine, but the activity of alkaline phosphatase was increased. Furthermore, activities of maltase and sucrase in the intestinal mucosa were elevated in streptozotocin-induced diabetic rats and were restored to control levels after dietary L-arginine supplementation. On the basis of the present experimental evidence, dietary L-arginine supplementation appears to affect the metabolism of lipoproteins and might alleviate some gastrointestinal dysfunctions, commonly seen in diabetes mellitus.