Dataset Information

Macromolecular and Solution Properties of the Recombinant Fusion Protein HUG.

ABSTRACT: The recombinant fusion protein HELP-UnaG (HUG) is a bifunctional product that exhibits human elastin-like polypeptide (HELP)-specific thermal behavior, defined as a reverse phase transition, and UnaG-specific bilirubin-dependent fluorescence emission. HUG provides an interesting model to understand how its two domains influence each other's properties. Turbidimetric, calorimetric, and light scattering measurements were used to determine different parameters for the reverse temperature transition and coacervation behavior. This shows that the UnaG domain has a measurable but limited effect on the thermal properties of HELP. Although the HELP domain decreased the affinity of UnaG for bilirubin, HUG retained the property of displacing bilirubin from bovine serum albumin and thus remains one of the strongest bilirubin-binding proteins known to date. These data demonstrate that HELP can be used to create new bifunctional fusion products that pave the way for expanded technological applications.

SUBMITTER: Sist P

PROVIDER: S-EPMC9364316 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Macromolecular and Solution Properties of the Recombinant Fusion Protein HUG.

Sist Paola P Bandiera Antonella A Urbani Ranieri R Passamonti Sabina S

Biomacromolecules 20220725 8

The recombinant fusion protein HELP-UnaG (HUG) is a bifunctional product that exhibits human elastin-like polypeptide (HELP)-specific thermal behavior, defined as a reverse phase transition, and UnaG-specific bilirubin-dependent fluorescence emission. HUG provides an interesting model to understand how its two domains influence each other's properties. Turbidimetric, calorimetric, and light scattering measurements were used to determine different parameters for the reverse temperature transition ...[more]

PMID: 35876275

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Project description:IntroductionProphylactic treatment of hemophilia B with recombinant factor IX (rFIX) molecules with enhanced pharmacokinetics including rIX-FP (albutrepenonacog alfa; Idelvion©) and rFIXFc (eftrenonacog alfa; Alprolix©) have commonly been used in the clinic. In the absence of head-to-head comparative trials, the aim of this study was to estimate the efficacy of rIX-FP versus rFIXFc using matching-adjusted indirect comparisons (MAICs).MethodsMAIC analyses leveraged individual patient data from the PROLONG-9FP trial and published summary-level data from the B-LONG trial for subjects who received weekly prophylaxis regimens. Individual patient data were used to assign weights and balance subjects from PROLONG-9FP with subjects from B-LONG on baseline disease severity, age, prior FIX regimen, and body mass index (BMI). Six efficacy outcomes were analyzed including annualized bleeding rate (ABR), annualized spontaneous bleeding rate (AsBR), annualized joint bleeding rate (AjBR), and the proportion of subjects without bleeding events (for total, spontaneous, and joint bleeding events).ResultsAfter adjustment for baseline disease severity, age, prior FIX regimen, and BMI, rIX-FP was associated with a statistically significant decrease in AsBR (rate ratio [RR] 0.42; 95% confidence interval [CI] 0.22, 0.82; P = 0.0107), and the proportion of patients without bleeding events (odds ratio [OR] 3.24; 95% CI 1.41, 7.45; P = 0.0057), spontaneous bleeding events (OR 3.47; 95% CI 1.56, 7.73; P = 0.0023), and joint bleeding events (OR 2.41; 95% CI 1.10, 5.26; P = 0.0274) compared with rFIXFc. Prophylactic treatment with rIX-FP was also associated with a numerically lower ABR (RR 0.75; 95% CI 0.32, 1.75; P = 0.5095) and AjBR (RR 0.82; 95% CI 0.37, 1.82; P = 0.6178).ConclusionThe MAICs demonstrated that weekly prophylaxis treatment of severe hemophilia B with rIX-FP resulted in favorable efficacy outcomes as compared to rFIXFc. These findings suggest rIX-FP may offer improved clinical benefits over rFIXFc.

Project description:BackgroundPlant lipid transfer proteins (LTPs) assemble a family of small (7-9 kDa) ubiquitous cationic proteins with an ability to bind and transport lipids as well as participate in various physiological processes including defense against phytopathogens. They also form one of the most clinically relevant classes of plant allergens. Nothing is known to date about correlation between lipid-binding and IgE-binding properties of LTPs. The garden pea Pisum sativum is widely consumed crop and important allergenic specie of the legume family. This work is aimed at isolation of a novel LTP from pea seeds and characterization of its structural, functional, and allergenic properties.ResultsThree novel lipid transfer proteins, designated as Ps-LTP1-3, were found in the garden pea Pisum sativum, their cDNA sequences were determined, and mRNA expression levels of all the three proteins were measured at different pea organs. Ps-LTP1 was isolated for the first time from the pea seeds, and its complete amino acid sequence was determined. The protein exhibits antifungal activity and is a membrane-active compound that causes a leakage from artificial liposomes. The protein binds various lipids including bioactive jasmonic acid. Spatial structure of the recombinant uniformly (13)C,(15)N-labelled Ps-LTP1 was solved by heteronuclear NMR spectroscopy. In solution the unliganded protein represents the mixture of two conformers (relative populations ~ 85:15) which are interconnected by exchange process with characteristic time ~ 100 ms. Hydrophobic residues of major conformer form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ~1000 Å(3)). The minor conformer probably corresponds to the protein with the partially collapsed internal cavity.ConclusionsFor the first time conformational heterogeneity in solution was shown for an unliganded plant lipid transfer protein. Heat denaturation profile and simulated gastrointestinal digestion assay showed that Ps-LTP1 displayed a high thermal and digestive proteolytic resistance proper for food allergens. The reported structural and immunological findings seem to describe Ps-LTP1 as potential cross-reactive allergen in LTP-sensitized patients, mostly Pru p 3(+) ones. Similarly to allergenic LTPs the potential IgE-binding epitope of Ps-LTP1 is located near the proposed entrance into internal cavity and could be involved in lipid-binding.

Dataset Information

Macromolecular and Solution Properties of the Recombinant Fusion Protein HUG.

Publications

Macromolecular and Solution Properties of the Recombinant Fusion Protein HUG.

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