Biomedical subjects

Ruth Freitag

Publications and source records attributed to Ruth Freitag.

22 records · Page 2Linked to original sources

PubMedDate not supplied

Continuous separation of multicomponent protein mixtures by annular displacement chromatography.

Displacement chromatography is a predominantly nonlinear mode of chromatography, which has certain advantages over the elution mode for preparative bioseparations. Whereas continuous production (and separation) processes have their theoretical benefits in this context, protein displacement chromatography has up to now only been performed in the batch mode. In this contribution, we demonstrate that the principle of continuous annular chromatography can be adapted to displacement chromatography. Separations of up to three standard proteins (two whey proteins, soybean trypsin inhibitor) were developed and optimized using a small (4 x 250 mm) batch column. These separations were subsequently transferred directly to the continuous system (500-mL column). Separations of similar quality in terms of final product purity and recovery yield were obtained using the continuous system.

Adsorption↗

PubMedDate not supplied

Capture of bacteria from fermentation broth by body feed filtration: a solved problem?

The direct capture of bacteria produced in high cell density fermentation by filtration is not possible once the milliliter-scale has been surpassed. Filtration in the presence of a filter aid (body feed filtration) constitutes a putative and scalable alternative, but only if conditions proposed by industry for large-scale filtration processes, namely, flow rates (for aqueous solutions) in the range of 500-1,500 L/(m(2) x h) and a filter aid concentration of <or=20 g/L, can be met. To this end several filter aids (grades of cellulose, diatomaceous earth, mixes thereof) were tested, albeit with no success. Capture and retention of bacteria was low (not possible in the case of cellulose), and the produced filter cakes were unstable and of low permeability. In the case of the diatomaceous earth close to 100% bacterial capture was possible, but only with filter aid concentrations that were 1 order of magnitude above the proposed limit. By using flocculating agents (Fe(3+)/Al(3+), PEI) either alone or in combination with diatomaceous earth and also the use of positively charged cellulose, bacteria capture could be achieved from several liters of cell suspension (OD(600) <or= 15) in filtration experiments adhering to the industrial recommendations. However, the bacteria were only weakly retained in such cakes and easily released by sudden pressure pulses. Satisfactory results were only obtained by filtration of the bacteria suspension at slightly alkaline pH (8.0, 50 mM Tris buffer) after treatment with sodium-activated bentonite (5 g/L) and PEI (50 mg/L) in the presence of 250 mM NaCl. In such cases 100% of the bacteria could reproducibly be captured in a filtration using 20 g/L of diatomaceous earth as filter aid. The thus produced filter cakes strongly retained the bacteria and showed good filtration performance. The procedure is at present limited to E. coli culture with biomass contents of OD(600) <or= 15. Cultures with higher OD(600) have to be diluted prior to filtration.

Bentonite↗

PubMedDate not supplied

Design and characterization of stimuli-responsive FLAG-tag analogues and the illumination-induced modulation of their interaction with antibody 4E11.

An azobenzene group containing beta-amino acid N-Fmoc-4-aminomethyl phenylazobenzoic acid was synthesized and with the exception of the C-terminal amino acid residue was substituted by solid-phase peptide synthesis into all positions of the FLAG sequence (DYKDDDDK), an octapeptide capable of specific interaction with the monoclonal antibody 4E11. The trans state of the beta-amino acid was thermodynamically more stable than the cis state. However, the molecule could be switched into the cis conformation by illumination at 340 nm. Peptides containing the artificial amino acid also became photoresponsive. In the absence of light, the spontaneous back-isomerization into the trans conformation of the photoresponsive was extremely slow (>8 h no significant increase in trans content). When illuminated with visible light (440 nm), the back-isomerization from the cis to the trans state was accelerated and occurred with a half-life of approximately 10 min. The cis form of the photopeptides was more hydrophilic than the trans form, as evidenced by differences in the retention time of the two isomeric forms in reversed-phase chromatography. Photopeptides that contained the intact sequences responsible for binding of the FLAG tag to the antibody, namely, the DYK motive at the N-terminus, showed binding to the antibody in both a dot blot immunoassay and in Biacore binding studies, albeit with lower affinity than the unmodified FLAG sequence. Peptides with a substitution in positions 4-6 showed differences in binding strength between the trans and the cis form in the Biacore studies, no such difference could be observed for the peptide with a substitution in position 7.

Antibodies, Monoclonal↗

PubMedDate not supplied

Purification of RT-PCR competent poly(A) mRNA from crude cell lysate by affinity precipitation.

Stimuli-responsive bioconjugates consisting of avidin covalently linked to poly(N-isopropylacrylamide) were used for the recovery of poly(A) mRNA hybridized to biotinylated poly(dT)-tags from crude cell lysates (Jurkat cells) by affinity precipitation. The bioconjugates are soluble in cold water but precipitate readily once a critical solution temperature (33 degrees C in pure water) is surpassed. The process is fully reversible and shows the expected dependencies on the composition of the aqueous solution and the bioconjugate chemistry. The results of the affinity precipitation were compared to those achieved with an accepted standard purification of poly(A) mRNA using avidin-activated magnetic beads. Both yield and quality/purity of the affinity precipitated poly(A) mRNA were found to be similar or better (especially removal of rRNA) than for poly(A) mRNA prepared by the magnetic particle-based protocol, while both mRNA isolates performed equally well in standard reverse transcriptase amplification (RT-PCR) of a beta actin transcript fragment. Poly(A) mRNA purification schemes based on affinity precipitation require no dedicated equipment and should have advantages in terms of scalability, handling, and costs.

Cell-Free System↗