Increasing the capacity of parvovirus-retentive membranes: performance of the Viresolve Prefilter.

Removal of small parvoviruses from highly purified proteins can be performed using normal-flow filters. The entrapment of protein aggregates, denatured proteins and other impurities can cause plugging and a decrease in filter capacity. In the present study a variety of prefilters were investigated for their ability to remove the species that foul Viresolvetrade mark NFP (normal-flow parvovirus) filters. The Viresolvetrade mark Prefilter, which utilizes entrapped diatomaceous earth to hydrophobically bind fouling species, provided a dramatic increase in virus filter capacity for solutions containing human IgG or a variety of monoclonal antibodies. We found that the component of the human IgG stream that bound to the Prefilter, when analysed using SDS/PAGE, isoelectric-focusing, size-exclusion chromatography, CD and ANS (1-anilinonaphthalene-8-sulphonate) titration, consisted of monomeric IgG variants containing more exposed hydrophobic surfaces. The bound component may represent oxidized or otherwise degraded IgG species or a subset of IgG molecules with more hydrophobic antigen-binding surfaces. The results indicate that NFP membranes do not foul solely as a result of entrapment of protein aggregates in the pore structure. The Viresolvetrade mark Prefilter has a high permeability, did not diminish protein yield and provided consistent performance between different media lots, device lots and device scales.

Computational and experimental probes of symmetry mismatches in the Arc repressor-DNA complex.

Arc repressor from bacteriophage P22 is a homodimeric member of the ribbon-helix-helix family of transcription factors. Two dimers bind cooperatively to adjacent sites on operator DNA with the antiparallel beta-sheet of each Arc dimer contacting the major groove. Despite the 2-fold symmetry of the dimer, the sequence of the DNA half-site is not symmetric, and thus there is an inherent symmetry mismatch in the complex. Here, using a combination of computational and experimental methods, we further characterize this symmetry mismatch. Continuum electrostatic calculations show that the DNA-contacting side-chains of Gln9, Asn11 and Arg13 each make better electrostatic interactions in one subunit of the Arc dimer than in the other. Based on the computational results, one or both Gln9 side-chains were substituted with Arg or His and Asn11 was similarly substituted with Ile in the context of a single-chain variant of Arc repressor. Although the double mutants that maintain the symmetry of the protein bind much more weakly than scArc, mutants with single Q9R or N11I substitutions bind operator DNA with affinities close to wild-type. Whereas the effects are nearly additive for the mutations at position 9, the effects of single N11I mutations are non-additive. The calculations thus enabled the identification of functionally tolerated mutations in the DNA-binding surface of Arc repressor, which in turn revealed energetic consequences of asymmetric environments in this protein-DNA interaction.

Expression of N-formylated proteins in Escherichia coli.

In bacteria, protein expression initiates with a formyl-methionine group. Addition of the antibiotic actinonin, a known peptide deformylase inhibitor, at the time of induction of protein expression results in the retention of the formyl group by the overexpressed protein. In addition, because deformylation is a prerequisite for removal of the initiating methionine, this post-translational processing step is also prevented by actinonin, and the N-formyl methionine residue is retained by proteins from which it is normally removed. We have demonstrated the applicability of this system for obtaining N-modified forms of several different proteins and use one of these modified molecules to show that the N-terminal amino group is not required for ClpXP degradation of proteins bearing an N-terminal recognition signal.