James C. Pearson

New strategy for the design of ligands for the purification of pharmaceutical proteins by affinity chromatography

A new approach for the identification of ligands for the purification of pharmaceutical proteins by affinity chromatography is described. The technique involves four steps. Selection of an appropriate site on the target protein, design of a complementary ligand compatible with the three-dimensional structure of the site, construction of a limited solid-phase combinatorial library of near-neighbour ligands and solution synthesis of the hit ligand, immobilisation, optimisation and application of the adsorbent for the purification of the target protein. This strategy is exemplified by the purification of a recombinant human insulin precursor (MI3) from a crude fermentation broth of Saccharomyces cerevisiae.

Design and application of bio-mimetic dyes in biotechnology.

The last decade or so has been the introduction of multi-coloured reactive dyes as substitutes for natural biological ligands in the purification of proteins by affinity chromatography. This paper reviews the evidence for the remarkable selectivity of the interaction of reactive dyes with proteins and describes our recent work with dye analogues. Terminal ring, bridging ring and anthraquinone ring analogues of Cibacron Blue F3G-A were synthesised de novo and shown to interact selectively with the NAD+-binding site of horse liver alcohol dehydrogenase but with affinities differing by several orders of magnitude. It is anticipated that these novel dye ligands will lead to affinity adsorbents with improved affinity, capacity and specificity.

Affinity precipitation of lactate dehydrogenase with a triazine dye derivative: selective precipitation of rabbit muscle lactate dehydrogenase with a procion blue H-B analog.

A simple methoxylated derivative of the triazine dye, Procion blue H-B, selectively precipitates rabbit muscle lactate dehydrogenase from solution. Optimum protein precipitation occurred at an enzyme subunit:dye ratio of approximately 2:1 and was fully reversible upon addition of competitive ligands such as NADH. With a crude extract of rabbit muscle, affinity precipitation with the dye followed by dissolution with NADH yielded homogeneous lactate dehydrogenase in 97% overall yield.

Preparative affinity precipitation of L-lactate dehydrogenase

Abstract The methoxylated p -sulphonate isomer of the triazine dye C.I. Reactive Blue 2, selectively precipitates L-lactate dehydrogenase from crude rabbit muscle extracts. At mildly alkaline pH values and a 7-fold molar excess of the dye analogue to enzyme subunits, 106 mg of homogeneous lactate dehydrogenase essentially free of soluble ligands and the principal contaminating enzyme activities, may be obtained in 60% overall yield from 100 g tissue in less than 3 h.

Biomimetic Dyes in Biotechnology

Downstream processing refers to all the technologies that are responsible for the production of pure products after fermentation. Therefore, if one were to produce, for example, a protein, one would start with an appropriate bio-reactor containing native or engineered cells. The first step would be to separate the cells from the broth; if the product is an intra-cellular one, one would subsequently disrupt the cells to release the intra-cellular products and then embark on a clarification process to obtain a clear extract containing the protein of interest. The next step is to apply a whole series of high resolution purification techniques, particularly chromatographic steps, prior to subsequently ending up with pure protein. Therefore, downstream processing entails the execution of primary recovery stages followed by a series of high-resolution steps where we add value to the final product and then hopefully end up with pure homogeneous protein. Special interest is focused in this report on the high resolution stages of the process leading to pure product and particularly those steps involving the most refined version of chromatography, affinity chromatography (1). The technique of affinity chromatography exploits small ligands which bind specifically and reversibly to the protein of interest. The appropriate small ligand is covalently attached to a suitable solid support matrix in such a way that we can establish that as a column.

Stable Synthetic Affinity Ligands for Use in Protein Fractionation

Stable synthetic affinity ligands such as reactive dyes have many advantages over biological ligands such as antibodies or cofactors when used in industrial protein fractionation processes. The properties of conventional reactive textile dyes can be improved by synthesising dye ligands with structures better suited to selective protein binding. Affinity adsorbents based on biomimetic dye ligands hold great promise for the biotechnology industry.