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Tissue Factor – A model protein

Group leader: Magdalena Svensson

Tissue Factor – A model protein for elucidation of single amino-acid contribution to β-sheet protein stability and stabilization achieved by specific additives

The number of recombinant proteins in clinical trials for new and existing therapeutic targets continues to increase each year. Despite the success of biotherapeutics there remain significant challenges to be overcome in maintaining product stability and efficacy throughout the production cycle and during long-term storage. Therefore, a deeper understanding of the contribution to the stability from each amino acid is deeply warranted.

The main model protein in these studies will be tissue factor (sTF), which has the advantage of being well characterized both structurally and functionally. sTF belongs to the cytokine receptor family. Members of this family are characterized by their fibronectin III (FNIII) domains, in which two antiparallel β-sheets, carrying three and four strands respectively, are packed together in a sandwich-like arrangement. sTF consists of two FNIII domains (Fig.1.).
Figure 1: Structure of Tissue factor with residues 74 and 188 indicated in red.

Antiparallel β-structures comprise the second largest group of protein domain structures. Functionally, this group is the most diverse; includes enzymes, transport proteins, antibodies, cell surface proteins, and virus coat proteins. Hence, our model protein of choice, sTF has the double advantage of being functionally well characterized as well as harbouring a very common protein fold.

In a host-guest study, one position in a protein is substituted for all of the naturally occurring amino acids and the stabilities of the protein variants are compared. This gives the propensities for the amino acids in the structure of choice and their contribution to the structural stability. The unique with sTF is that two equivalent positions in sTF can be chosen as guest sites (for example R74 and S188, one at a time), and will thereby serve as an internal reference for each other.

Recently, we have extended our studies on sTF and mutants thereof to include the effect on stability caused by additives. A basic goal of biopharmaceutical formulations is to enhance the stability of the protein product to maintain its efficiency during storage and transportation. The development of an effective recipe for formulation would be improved by understanding of the physico-chemical principles that govern protein stability.

Ever since biological scientists began isolating proteins there has been a struggle to stabilize proteins with solvent additives. Timasheff and co-workers (S.N. Timasheff (1998) Adv. Protein Chem, 51: 355-432) have elucidated a thermodynamic mechanism for enhancement of protein stability induced by solvent additives. The general observation has been that those cosolvents that stabilize protein structure are preferentially excluded from the protein surface, whereas those that induce unfolding in general interact favorably with the unfolded state of the protein.

However, it is still not today possible to estimate beforehand the amount of stabilization achieved by a specific additive. The methods developed are on an individual basis for each specific protein [45]. With our double- model protein at hand we have an exceptional possibility to discriminate between global and local effects of the additives.

Thus, fundamental studies of the effects of solution conditions on protein stability will provide us with guiding principles for stabilization strategies and eliminate the need for case-by-case stabilization. The final objective is to find biopharmaceutical formulations that will prolong the shelf-life of the proteins.

Responsible for this page: Maria Sunnerhagen
Last updated: 05/27/08