Validation, Deployment, and Real-World Implementation of a Modular Toolbox for Alzheimer's Disease Detection and Dementia Risk Reduction: The AD-RIDDLE Project.

The Real-World Implementation, Deployment, and Validation of Early Detection Tools and Lifestyle Enhancement (AD-RIDDLE) project, recently launched with the support of the EU Innovative Health Initiative (IHI) public-private partnership and UK Research and Innovation (UKRI), aims to develop, test, and deploy a modular toolbox platform that can reduce existing barriers to the timely detection, and therapeutic approaches in Alzheimer's disease (AD), thus accelerating AD innovation. By focusing on health system and health worker practices, AD-RIDDLE seeks to improve and smooth AD management at and between each key step of the clinical pathway and across the disease continuum, from at-risk asymptomatic stages to early symptomatic ones. This includes innovation and improvement in AD awareness, risk reduction and prevention, detection, diagnosis, and intervention. The 24 partners in the AD-RIDDLE interdisciplinary consortium will develop and test the AD-RIDDLE toolbox platform and its components individually and in combination in six European countries. Expected results from this cross-sectoral research collaboration include tools for earlier detection and accurate diagnosis; validated, novel digital cognitive and blood-based biomarkers; and improved access to individualized preventative interventions (including multimodal interventions and symptomatic/disease-modifying therapies) across diverse populations, within the framework of precision medicine. Overall, AD-RIDDLE toolbox platform will advance management of AD, improving outcomes for patients and their families, and reducing costs.

Fusion proteins in biotechnology.

Gene fusion techniques allow the production of recombinant proteins featuring the combined characteristics of the parental products. Originally, these techniques were used to probe transcriptional and translational activity, to translocate proteins across cell membranes, and to facilitate the recovery of proteins. Recently, new applications have emerged in areas such as protein refolding, immunology, drug targeting and protein display. A slightly modified version of this review is also published in Current Opinion in Structural Biology 1992, 2:569-575.

Isolation and characterization of two biologically active O-glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae. Identification of a new motif for O-glycosylation.

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the O-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O-glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C-terminal O-glycosylation of hPTH with up to seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I.

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1-3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1-3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1-3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.

Disulfide exchange folding of insulin-like growth factor I.

The disulfide exchange folding properties of insulin-like growth factor I (IGF-I) have been analyzed in a redox buffer containing reduced (10 mM) and oxidized (1 mM) glutathione. Under these conditions, the 3 disulfide bridges of the 70 amino acid peptide were not quantitatively formed. Instead, five major forms of IGF-I were detected, and these components were concluded to be in equilibrium as their relative amounts were similar starting from either reduced, native, or a mismatched variant of IGF-I containing two non-native disulfides. The different components in the mixtures were trapped by thiol alkylation using vinylpyridine and subsequently isolated by reverse-phase HPLC. The purified variants were further characterized using plasma desorption mass spectrometry and peptide mapping. Two of the five different forms were identified as native and mismatched IGF-I. One form was a variant with only one disulfide bond, and the other two major components had two disulfides formed. In a separate experiment, early refolding intermediates were trapped by pyridylethylation after only 90 s of refolding in the glutathione buffer, starting from reduced IGF-I. The intermediates were identical to the components observed at equilibrium, but at different relative concentrations. On the basis of the disulfide bond patterns of the different components in the equilibrium mixtures, we conclude that the disulfide between cysteines-47 and -52 in IGF-I is an unfavorable high-energy bond that may exist in the native molecule in a strained configuration.

Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone.

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly in Escherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzymes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.

Purification and characterization of recombinant human insulin-like growth factor II (IGF-II) expressed as a secreted fusion protein in Escherichia coli.

Human insulin-like growth factor II (IGF-II) was produced in an Escherichia coli ompT strain as a 22.5-kDa fusion protein. IGF-II was fused to the carboxy-terminal of a synthetic 15-kDa IgG-binding protein, originating from staphylococcal protein A, via a unique methionine linker. During fermentation, the fusion protein was exported to the growth medium at levels exceeding 900 mg/liter and subsequently affinity purified on IgG Sepharose followed by ion exchange on S Sepharose. After chemical cleavage with CNBr, yielding an authentic IGF-II molecule, the recombinant IGF-II was purified to homogeneity by a two step procedure involving ion-exchange and reverse-phase HPLC. A substantial fraction of the secreted protein was found to be biologically active, eliminating the need for complex refolding procedures. The yield of highly purified and biologically active IGF-II was 5-7 mg/liter of fermenter broth. The IGF-II produced by this method displayed biochemical, immunological, receptor binding, and biological activity properties equal to those of native IGF-II isolated from human serum.

Characterization of an extended form of recombinant human insulin-like growth factor II.

To investigate the biological role of variants of human insulin-like growth factor II (IGF-II), an extended form designated IGF-IIE21, with a molecular mass of 9.8 kDa, was produced in Escherichia coli as a stable and soluble secreted fusion protein. After site-specific cleavage of the affinity purified fusion protein, followed by purification using ion exchange and reversed phase chromatography, it could be demonstrated that IGF-IIE21 and IGF-II have similar or identical activities according to radioimmunoassay and radioreceptor assay. However, IGF-IIE21 showed only 1% growth promotion activity as compared with IGF-II in a clonal expansion assay using human K562 cells which lacks IGF-I receptors. These results suggest that this extended variant of IGF-II can bind to the receptor but has limited growth promoting activity.

Facilitated in vitro refolding of human recombinant insulin-like growth factor I using a solubilizing fusion partner.

We describe a new approach to refolding recombinant proteins in which an affinity fusion partner, consisting of two IgG-binding domains (ZZ) derived from staphylococcal protein A, is used to solubilize misfolded molecules before, during and after reduction and reoxidation. We show that human insulin-like growth factor I (IGF-I) can be refolded as a fusion protein at a concentration as high as 1-2 mg/ml without the use of denaturing agents. A process scheme suitable for large scale application is described in which the yield of correctly folded human IGF-I with full biological activity is substantially increased.

Human immunodeficiency viral protease is catalytically active as a fusion protein: characterization of the fusion and native enzymes produced in Escherichia coli.

Processing of the gag and pol gene precursor proteins of retroviruses is essential for the production of mature infectious virions. The processing is directed by a viral protease that itself is part of these precursors and is presumed to cleave itself autocatalytically. To facilitate study of this process, the protease was produced as a fusion protein in Escherichia coli. In this construct, the 10,793-Da protease was preceeded by two copies of a modified IgG binding domain derived from protein A. The IgG binding domain was linked to the protease by an Asp-Pro peptide bond which could not be cleaved by the viral protease. A dimer of the 25,400-Da fusion protein was catalytically active, specifically cleaving a substrate peptide at the correct Tyr-Pro bond. Thus, the fusion protein could serve as a model of the viral gag-pol polyprotein. The finding that the fusion protein was catalytically active supports the suggestion that a gag-pol dimer can initiate a proteolytic cascade after budding of the immature virus. The fusion protein also provided a source of authentic protease. The protease was released from the fusion construct by incubation with formic acid, cleaving the Asp-Pro linkage which had been inserted between the IgG binding domain and the protease.

Separation and characterization of modified variants of recombinant human insulin-like growth factor I derived from a fusion protein secreted from Escherichia coli.

Human insulin-like growth factor I, IGF-I, was produced in Escherichia coli fused to a synthetic IgG-binding peptide The fusion protein is secreted into the medium during fermentation and was initially purified on an IgG-Sepharose column. After hydroxylamine cleavage, IGF-I was purified to homogeneity. During purification, impurities in the form of modified variants of IGF-I were detected and characterized. The closely related impurities were identified to be a misfolded form of IGF-I, having mismatched disulphide bonds, a form with the single methionine residue in IGF-I oxidized to methionine sulphoxide and a variant in which the methionine residue was substituted by a norleucine residue during protein synthesis. A form proteolytically cleaved between two arginine residue was also detected. These impurities were separated from the major component, native IGF-I, by using reverse-phase h.p.l.c. The modified molecules as well as native IGF-I were characterized both as intact molecules and as fragments, after pepsin digestion, using the techniques of plasma desorption m.s., N-terminal sequencing and amino acid analysis. The oxidized form was 90%, and the norleucine analogue was 70%, as potent as native IGF-I in a biological radioreceptor assay, and the form having mismatched disulphides lacked receptor affinity.

Expression and characterization of a recombinant human parathyroid hormone secreted by Escherichia coli employing the staphylococcal protein A promoter and signal sequence.

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids (hPTH(1-84)). Employing the promoter and signal sequence of Staphylococcus aureus-protein A we have expressed hPTH in Escherichia coli. The expressed proteins are excreted to the growth medium, allowing for rapid and easy purification of the desired products. By amino acid sequence analysis and mass spectrometry, we have shown that the major excreted product is correctly processed human identical hPTH(1-84). The purified recombinant hPTH(1-84) stimulates adenylate cyclase activity in rat osteosarcoma cell membranes to exactly the same extent as synthetic parathyroid hormone standards, indicating that the recombinant product has full biological activity.

Comparison of two chemical cleavage methods for preparation of a truncated form of recombinant human insulin-like growth factor I from a secreted fusion protein.

We have produced a naturally occurring variant of human insulin-like growth factor I, truncated by three amino acids at the amino terminus. The polypeptide is obtained as a fusion protein in Escherichia coli. The fusion partner is a synthetic IgG-binding peptide. During fermentation the fusion protein is secreted into the medium, and is purified on IgG--Sepharose prior to cleavage. Two different genes for the fusion protein were used, allowing chemical cleavage at either a tryptophan linker or a methionine linker between the fusion partner and the growth factor, using N-chlorosuccinimide (NCS) or cyanogen bromide (CNBr) respectively. A partial CNBr cleavage yielded the native peptide, whereas the NCS cleavage yielded a product in which the single methionine had been oxidized to the sulfoxide. The forms from both cleavage methods exhibited biological activity and were characterized after purification to homogeneity. Both cleavage methods gave products having correct N- and C-terminal ends. The purified product had a biological activity equal to that of corresponding material from natural sources, 15 000 U/mg. Modified forms of truncated IGF-I were also identified, purified and characterized. Modifications such as proteolysis and misincorporation of norleucine for methionine occurred during biosynthesis, while oxidation of methionine took place during both fermentation and chemical cleavage.

A comparison of the biological activity of the recombinant intact and truncated insulin-like growth factor 1 (IGF-1).

A truncated form of insulin-like growth factor 1 (IGF-1), which lacked the aminoterminal tripeptide Gly-Pro-Glu has been isolated from human fetal and adult brain. This truncated IGF-1 displayed more potent cross-reactivity and biological action on brain cells than IGF-1 isolated from human serum. We now present data on a recombinant DNA-derived truncated IGF-1 lacking the aminoterminal tripeptide. Recombinant truncated IGF-1 was 1.4-5-times more potent than recombinant and natural IGF-1 in displacing [125 I]IGF-1 from human fetal and adult brain and placenta membranes. These differences were slightly enhanced when truncated IGF-1 was used as radioligand. The relative potencies compared to insulin-like growth factor 2 (IGF-2) in displacing [125I]IGF-2 from rat liver membranes were recombinant truncated IGF-1, 0.3% and recombinant IGF-1, 0.2%. Recombinant truncated IGF-1 displayed 100-fold reduced affinity for the low molecular weight binding protein (IGF-BP) isolated from human amniotic fluid when compared to recombinant IGF-1. Likewise, the IGF-BP was 100-fold less potent in inhibiting the receptor binding of recombinant truncated IGF-1 than that of recombinant IGF-1. Recombinant truncated IGF-1 was 4-times more potent than recombinant and natural IGF-1 in stimulating DNA synthesis in fetal rat brain cells. This biological activity of recombinant truncated IGF-1 was not affected by the IGF-BP at concentrations which abolished the biological activity of recombinant IGF-1. The hypothesis that IGF-BP bound intact IGF-1 represents the endocrine form of IGF-1, whereas truncated IGF-1 represents the paracrine or autocrine form of IGF-1, is proposed.

Solubilization of a membrane-bound diol dehydratase with retention of EPR g = 2.02 signal by using 2-(N-cyclohexylamino)ethanesulfonic acid buffer.

A procedure for solubilization of the oxygen-labile, membrane-bound diol dehydratase from Clostridium glycolicum with retention of enzymatic activity is described. The procedure involves sonication of crude membrane preparations anaerobically in 0.1 M 2-(N-cyclohexylamino)ethane-sulfonic acid (CHES) buffer (pH 8.6-9.0) containing 2 mM dithiothreitol. The addition of dimethylsulfoxide (30%) and lysophosphatidylcholine (0.15 mg/ml) to the solubilization buffer resulted in a 10-fold increase in recovery of solubilized diol dehydratase activity. After ultracentrifugation, an overall recovery of 50% of the activity initially present in the crude membrane preparations was achieved. Active membrane preparations and the solubilized enzyme exhibited an EPR signal at g = 2.02. Both enzyme activity and EPR signal were sensitive to oxygen and the radical scavengers, NH2OH and hydroxy-urea.

Butyrate kinase from Clostridium acetobutylicum.

Crude extracts of Clostridium acetobutylicum contain a butyrate kinase of high specific activity (5.2 mumol/min/mg of protein). The enzyme has been purified 77-fold in a six-step procedure to a specific activity of 402 mumol/min/mg of protein. The purified butyrate kinase showed a single band with a molecular weight of 85,000 on nondenaturing polyacrylamide gradient gel electrophoresis. Separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme is a dimer of two apparently identical subunits with molecular weights of 39,000. The pH optimum for the reaction in the butyryl phosphate-forming direction is 7.5, and the pI of the kinase is 5.6. The amino acid composition of the enzyme is also reported. It contains no tryptophan and is low in sulfur-containing amino acids. The kinase has a broad substrate specificity and exhibits its highest relative activities with butyrate and valerate. Butyrate kinase is rapidly inactivated at 50 degrees C in the absence of a fatty acid substrate. Although a reducing agent was required for maximum activity, treatment with several sulfhydryl-modifying agents failed to inhibit the enzyme.

Diol metabolism and diol dehydratase in Clostridium glycolicum.

Levels of the five enzymes involved in the fermentation of 1,2-ethanediol and 1,2-propanediol in the strictly anaerobic bacterium, Clostridium glycolicum, were investigated. All enzymes with the exception of the first enzyme in the pathway, diol dehydratase, were found to be constitutive, stable to exposure to oxygen, and present in the cytosol. Diol dehydratase was found to be extremely oxygen sensitive and strongly associated with the cell membrane. Treatment with ionic and nonionic detergents, butanol, phospholipase A2, or osmotic shock procedures failed to solubilize any diol dehydratase activity. Limited proteolysis using subtilisin released small amounts of activity. Diol dehydratase was found to be specific for 1,2-ethanediol and 1,2-propanediol and required the addition of a reducing agent for maximal activity. The enzyme was strongly inhibited by low concentrations of EDTA, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, o-phenanthroline, hydroxylamine, hydroxyurea, and sulfhydryl reagents. Addition of adenosylcobalamin or high levels of intrinsic factor did not affect the reaction rate. Irradiation with light also did not inhibit the enzyme activity. These results suggest that the catalytic mechanism of diol dehydratase from C. glycolicum does not involve a cobamide coenzyme.

Simultaneous single-step purification of thiolase and NADP-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri.

Thiolase and NADP-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri were purified by ion-exchange chromatography to near homogeneity in a simultaneous, single-step procedure. The yield of both enzymes was greater than 80%. Thiolase was purified approximately 8-fold with sp act 115 units/mg, whereas 3-hydroxybutyryl-CoA dehydrogenase was purified 14-fold with sp act 292 units/mg. Isoelectric points of the enzymes are 7.7 for thiolase and 7.8 for 3-hydroxybutyryl-CoA dehydrogenase. Milligram quantities of each of these enzymes are readily obtained from this fatty acid-producing organism.