Novel in situ polymerized coatings for hydrophobic interaction chromatography media.

Hydrophobic interaction chromatography (HIC) and other capture media are typically produced by grafting different ligands to base matrices at defined surface densities. This often complicates media production. An alternative approach to media involving in situ radical initiated polymerization was used to graft polymer coatings directly at Sepharose(R) polymeric base matrices.

Biological Trojan horse: Antigen 43 provides specific bacterial uptake and survival in human neutrophils.

Escherichia coli is a versatile pathogen causing millions of infections in humans every year. This bacterium can form multicellular aggregates when it expresses a self-associating protein, antigen 43 (Ag43), on its surface. We have discovered that Ag43-expressing E.

Hydrophobic peptide tags as tools in bioseparation.

Hydrophobic interactions are highly selective, and differences in surface hydrophobicities between proteins can be used as an efficient handle to facilitate protein isolation. Aromatic amino acid residues are of particular importance for molecular recognition because they have a key role in several biological functions. The hydrophobicity of a protein can easily be altered with minor genetic modifications, such as site-directed mutagenesis or fusions of hydrophobic peptide tags.

N-terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition.

The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli.

Partitioning and characterization of tyrosine-tagged green fluorescent proteins in aqueous two-phase systems.

The green fluorescent protein GFPuv has been genetically engineered to investigate the influence of N-terminal tyrosine extensions in aqueous two-phase systems. Fusions in the N-terminus affected the protein expression, and tags containing three tyrosines and prolines influenced the expression favorably. This effect is probably due to changes in mRNA stability, because the amounts of corresponding mRNAs correlated with the amounts of GFPuv proteins.

Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns.

Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylamino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 microm in size.

Improved partitioning in aqueous two-phase system of tyrosine-tagged recombinant lactate dehydrogenase.

The partitioning of Bacillus stearothermophilus lactate dehydrogenase (LDH) in an aqueous two-phase system was studied. Particularly, the influence of tyrosine tags on the partitioning was evaluated. The hydrophobic effect, caused by the addition of tyrosine residues, was determined in a system based on dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer (EO30PO70).