Lars Skriver

Characterization of the three 125I-iodination isomers of human insulin-like growth factor I (IGF1)

Human insulin-like growth factor I (IGF1) was labeled with 125I and the resulting mixture of iodination isomers was separated by reverse-phase HPLC. Three major radioactive peaks were isolated and identified by sequencing as the expected three monoiodinated species. The ranking of the affinities of the three isomers for the human IGF1 receptor was found to be Tyr24(125I) > Tyr31(125I) >> Tyr60(125I). The Tyr31(125I) isomer was shown to have an affinity similar to that of unlabeled IGF1 and is thus the tracer of choice for IGF1. The tracers were stable upon storage at -20 degrees C for at least 3 months.

Stabilization of Recombinantly Expressed Proteins

Downstream purification of recombinant proteins has become an increasingly challenging issue. Even very fragile and complex proteins are now expressed in heterologous systems, and the successive purification processes are subject to still higher demands of process control and documentation. Usually, downstream purification involves multiple, highly technological and rather complicated unit operations in which the proteins are subject to an environment differing greatly from what can be found in vivo. The protein structure is sensitive to such environmental changes, and protein stability becomes a crucial aspect of protein purification. Therefore, state-of-the-art purification strategies should focus not only on the highest homogeneity and purity, but also on the presence of unstable forms of the protein. Stability problems are sometimes amplified in the downstream purification process, because the proteins are produced in a nonnative environment. Three very common causes of the emergence of stability problems could be listed as follows: Recombinant proteins are usually expressed in hcterologous systems; obviously, the required folding and post-translational modifications must be performed by enzyme systems that d o not exactly represent those found in the original in vivo host system. Stabilizing environment found in vivo might not be present during downstream purification. Such stabilizing factors could be redox potentials, binding proteins, folding enzymes, cellular compartments, chaperons, stabilizing cofactors, membranes, and so forth. 3. During expression and purification, the protein concentration is usually much above the in vivo concentration. Thus, intermolecular interactions might influence the stability dramatically. This paper exemplifies how stability problems might be solved by an optimal design of the purification process. 1 .