Molecular engineering of insulin for recombinant expression in yeast.

Since the first administration of insulin to a person with diabetes in 1922, scientific contributions from academia and industry have improved insulin therapy and access. The pharmaceutical need for insulin is now more than 40 tons annually, half of which is produced by recombinant secretory expression in Saccharomyces cerevisiae. We discuss how, in this yeast species, adaptation of insulin precursors by removable structural elements is pivotal for efficient secretory expression. The technologies reviewed have been implemented at industrial scale and are seminal for the supply of human insulin and insulin analogues to people with diabetes now and in the future. Engineering of a target protein with removable structural elements may provide a general approach to yield optimisation.

Structures of insect Imp-L2 suggest an alternative strategy for regulating the bioavailability of insulin-like hormones.

The insulin/insulin-like growth factor signalling axis is an evolutionary ancient and highly conserved hormonal system involved in the regulation of metabolism, growth and lifespan in animals. Human insulin is stored in the pancreas, while insulin-like growth factor-1 (IGF-1) is maintained in blood in complexes with IGF-binding proteins (IGFBP1-6). Insect insulin-like polypeptide binding proteins (IBPs) have been considered as IGFBP-like structural and functional homologues. Here, we report structures of the Drosophila IBP Imp-L2 in its free form and bound to Drosophila insulin-like peptide 5 and human IGF-1. Imp-L2 contains two immunoglobulin-like fold domains and its architecture is unrelated to human IGFBPs, suggesting a distinct strategy for bioavailability regulation of insulin-like hormones. Similar hormone binding modes may exist in other insect vectors, as the IBP sequences are highly conserved. Therefore, these findings may open research routes towards a rational interference of transmission of diseases such as malaria, dengue and yellow fevers.

Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers.

The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies.

Structural and biological properties of the Drosophila insulin-like peptide 5 show evolutionary conservation.

We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å. DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel ß-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel ß-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.

Coagulation factor XIII variants with altered thrombin activation rates.

Coagulation factor XIII (FXIII) is activated by thrombin and catalyses crosslinking between fibrin monomers thereby providing mechanical strength to the fibrin network. V34L is a common FXIII-A polymorphism found in the activation peptide. FXIII-A V34L is activated faster by thrombin and provides formation of a tighter clot at fibrinogen concentrations in the low end of the physiological range. FXIII-A variants with potentially increased activation rates were generated. Introduction of an optimal thrombin cleavage site, V34L+V35T, increased the activation rate 7.6-fold and facilitated the formation of a fibrin network more resistant to fibrinolysis than obtained with wt FXIII-A. In contrast, introduction of fragments of fibrinopeptide A into the activation peptide resulted in severely impaired activation rates.

IGF-I binding to the IGF-I receptor is affected by contaminants in commercial BSA: the contaminants are proteins with IGF-I binding properties.

OBJECTIVE: To determine whether different albumins have an effect on IGF-I binding assays. METHODS: We have studied the effect of five different albumins in plate antibody capture binding assay. For IGF-IR studies the IGF-IR specific antibody 24-31 was used and for IR/IGF-IR hybrid receptors the IR specific antibody 83-7 was used. Binding to IGF-IR was studied by displacement of (125)I-IGF-I with IGF-I in the absence or presence of 0.1%, 0.5% or 1% (w/v) albumin. The IR/IGF-IR hybrid receptors were studied in the presence of 0.5% (w/v) of HSA A-1887 and BSA A-7888 and with IGF-I or insulin displacement of (125)I-IGF-I. The albumins used were purchased from Sigma-Aldrich. Two batches of albumins from each catalog number were tested. The albumins were: HSA A-1887, BSA A-4503, BSA A-6003, BSA A-7030, and BSA A-7888. Contaminants in the albumins were characterized as proteins with IGF-I binding properties by cross-linking to (125)I-IGF-I and SDS-page analysis. RESULTS: BSA A-4503, A-7030 and A-7888 from Sigma-Aldrich contain proteins with IGF-I binding properties. These contaminants increased the determined EC50 for displacement of (125)I-IGF-I from IGF-IR up to 40-fold in a BSA dependent manner. The presence of BSA-7888 in binding experiments increased the determined EC50 for IR/IGF-IR hybrid receptors 8-16-fold. CONCLUSIONS: When IGF-I is characterized with respect to the effect on living cells and on binding to potential receptors unspecific binding to surfaces is often prevented by the addition of albumin in the assay. Here we report that when binding to the classical IGF-IR and IR/IGF-IR hybrid receptors are studied the measured EC50 values can be albumin dependent if it is contaminated with proteins with IGF-I binding properties. The free IGF-I concentration will be lower than estimated. Thus, the contaminated BSA preparations result in artifacts leading to misinterpretations and underestimation of the effect of IGF-I. Our results provide one possible explanation as to why different laboratories report different EC50 values for IGF-I.

Hybrid receptors formed by insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) have low insulin and high IGF-1 affinity irrespective of the IR splice variant.

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.

Functional reconstitution of insulin receptor binding site from non-binding receptor fragments.

We have previously shown that a minimized insulin receptor (IR) consisting of the first 468 amino acids of the insulin receptor fused to 16 amino acids from the C terminus of the alpha-subunit (CT domain) bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Schäffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780-17786). In the present study, we show that a smaller construct that has the first 308 residues fused to the CT domain also binds insulin. Insulin receptor fragments consisting of the first 468 or 308 residues did not bind insulin. However, when these fragments were mixed with a synthetic peptide corresponding to the CT domain, insulin binding was detectable. At concentrations of 10 microm CT peptide, insulin binding was fully reconstituted yielding apparent affinities of 9-11 nm. To further investigate the minimum requirement for the length of the N terminus of IR, we tested smaller receptor fragments for insulin binding in the presence of the CT peptide and found that a fragment consisting of the first 255 amino acids of IR was able to fully reconstitute the insulin binding site, yielding an apparent affinity of 11 +/- 4 nm for insulin.