A rapid, inexpensive method for producing water-soluble enzyme-coated micro-crystals which exhibit dramatically enhanced catalytic activity and stability in non-aqueous media and can be re-dissolved easily in aqueous solution is described.

Protein-coated microcrystals (PCMC), a biocatalyst preparation previously demonstrated to display substantially increased transesterification activity of proteases and lipases in organic solvents when compared to their as received counterparts [Kreiner M, Moore BD, Parker MC (2001) Chem. Commun. 12:1096--1097], was applied to oxidoreductases. Horse liver alcohol dehydrogenase (HLADH), catalase (CAT), soybean peroxidase and horseradish peroxidase were immobilised onto K(2)SO(4) crystals and dehydrated in a 1-step process, resulting in PCMC. These PCMC preparations showed enhanced activity in different organic solvents in most types of reactions tested. The highest activation was observed with HLADH (50-fold as active as enzyme as received) and CAT (25-fold).

Substrates utilising clustered arginine-glycine-aspartic acid (RGD) ligand displays support greater cell adhesion over random displays. However, cell adhesion to integrin alpha5beta1 requires the synergy site on the 9th type III fibronectin domain (FIII) in addition to RGD on the 10th FIII domain. Here, we have designed and expressed soluble protein chimeras consisting of an N-terminal 9th-10th FIII domain pair, IgG-derived hinge and leucine zipper-derived helix; the latter mutated to yield di-, tri- and tetrameric coiled coils and thus self-assembling, multimeric integrin alpha5beta1 ligands. A unique C-terminal cysteine was appended to the helix to facilitate 'anchoring' of the chimeras with a defined orientation on a surface. Size-exclusion chromatography and circular dichroism demonstrated that the chimeras self-assembled as multimers in solution with defined secondary structures predicted from theoretical calculations. Biotinylation via a thioether bond was used to selectively bind the chimeras to streptavidin-coated surfaces, each of which was then shown to bind integrin alpha5beta1 by surface plasmon resonance. Spreading of fibroblasts to surfaces derivatised with the chimeras was found to proceed in the order: tetramer > trimer > dimer > monomer. Thus, we describe novel polyvalent integrin alpha5beta1 ligands for facile derivatisation of substrates to improve cell adhesion in vitro.

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