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A multifaceted strategy to improve recombinant expression and structural characterisation of a Trypanosoma invariant surface protein.

ABSTRACT: Identification of a protein minimal fragment amenable to crystallisation can be time- and labour intensive especially if large amounts are required and the protein has a complex fold and functionally important post-translational modifications. In addition, a lack of homologues and structural information can further complicate the design of a minimal expression construct. Recombinant expression in E. coli promises high yields, low costs and fast turnover times, but falls short for many extracellular, eukaryotic proteins. Eukaryotic expression systems provide an alternative but are costly, slow and require special handling and equipment. Using a member of a structurally uncharacterized, eukaryotic receptor family as an example we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) guided construct design in conjunction with truncation scanning and targeted expression host switching to identify a minimal expression construct that can be produced with high yields and moderate costs.

SUBMITTER: Sulzen H

PROVIDER: S-EPMC9325691 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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A multifaceted strategy to improve recombinant expression and structural characterisation of a Trypanosoma invariant surface protein.

Sülzen Hagen H Votrubova Jitka J Dhillon Arun A Zoll Sebastian S

Scientific reports 20220726 1

Identification of a protein minimal fragment amenable to crystallisation can be time- and labour intensive especially if large amounts are required and the protein has a complex fold and functionally important post-translational modifications. In addition, a lack of homologues and structural information can further complicate the design of a minimal expression construct. Recombinant expression in E. coli promises high yields, low costs and fast turnover times, but falls short for many extracellu ...[more]

PMID: 35882923

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Project description:BACKGROUND:Human metapneumovirus (HMPV) has been shown to cause respiratory infection, accounting for approximately 7% of all such disease, and contributes to the development of asthma in humans. HMPV has a worldwide distribution with infectivity rates approaching 100%, and immunocompromised patients are particularly at risk from viral exposure. No anti-HMPV vaccine is available and diagnosis is primarily based on in-house molecular or serological tests, in part due to limited availability of recombinant HMPV antigens. OBJECTIVE:To generate a panel of HMPV-derived recombinant antigens, develop standardised ELISA systems for HMPV IgG detection and explore the nature of B cell memory against HMPV to underpin future vaccine studies. STUDY DESIGN:HMPV viral RNA was isolated from a clinical specimen and RT-PCR was conducted. The HMPV M and P genes were cloned and expressed in Escherichia coli. The HMPV N gene was cloned and expressed in insect cells using the baculovirus expression system. Each purified recombinant antigens was subsequently employed in HMPV-specific ELISA. RESULTS:High-level expression, and purification, of both HMPV matrix (M) (10 mg/g cells) and phosphoprotein (P) (3.82 mg/g cells) were achieved in an E. coli expression system. Recombinant HMPV (N) was successfully expressed in, and purified from the baculovirus expression system. Overall, a 99% HMPV IgG seroprevalence was observed (n = 96) using HMPV M-, N- and P-ELISA, respectively. The M antigen proved to be the most diagnostically useful with 99% of specimens tested exhibiting anti-M protein reactivity. A high correlation was observed between anti-M and N IgG reactivity (r = 0.96), with significant correlation also evident for anti-N and P IgG reactivity (r = 0.74). Lowest correlation was evident for anti-M and P IgG reactivity (r = 0.57). Finally, the first demonstration of HMPV-specific B cell memory (ranging 1-15 spot forming cells (SFC)/million cells) was achieved against M and P antigens in 40% of individuals tested. CONCLUSION:This work describes robust diagnostic systems for HMPV and new insight into antigen-specific B cell memory against HMPV.

Project description:ObjectiveThe U.S. Centers for Disease Control and Prevention encourages nurses to evaluate penicillin allergies as part of hospital-based antibiotic stewardship programs. We evaluated the feasibility of an implementation strategy to improve nurses' comprehensive documentation of penicillin allergies. We defined feasibility as the uptake and acceptability of documentation procedures.DesignSix-month pre-post feasibility implementation study.SettingOutpatient surgical areas of an academic medical center located in the U.S.InterventionThe implementation strategy was guided by the Capability, Opportunity, Motivation Model for Behavior Change and included, building an interdisciplinary coalition to iteratively evaluate the implementation effort, educational meetings with surgical prescribers and perioperative nurses, the development and distribution of educational pocket cards, and structured communication messages in the electronic medical record.ResultsA total of 426 patients with 487 penicillin allergy records (216 records pre-implementation period, 271 records post-implementation period) were analyzed. Penicillin allergy documentation contained the following information in the pre- versus post-implementation period: symptoms of the reaction (87% vs 87%), timing/years since reaction (8% vs 26%), onset of reaction in relation to taking penicillin (0% vs 21%), how symptoms resolved (0% vs 21%), and penicillin re-exposure (3% vs 21%). Focus groups revealed nurses perceived documentation procedures as highly acceptable. Major drivers of acceptability included the perceived effectiveness of a detailed allergy history and self-efficacy in conducting a detailed allergy history.ConclusionsNurses perceived the comprehensive documentation of penicillin allergy history intervention as acceptable, and uptake improved following a theory-informed implementation strategy. We offer implementation strategy components to facilitate nurses' engagement in penicillin allergy evaluation.

Dataset Information

A multifaceted strategy to improve recombinant expression and structural characterisation of a Trypanosoma invariant surface protein.

Publications

A multifaceted strategy to improve recombinant expression and structural characterisation of a Trypanosoma invariant surface protein.

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