Dataset Information

Mycobacterium tuberculosis protein production pipeline

ABSTRACT: Purpose: Tuberculosis is still a major threat to global health. New tools and strategies to produce disease-related proteins are quintessential for the development of novel vaccines and diagnostic markers. Experimental design: To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. The potential for the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays. Results: Twelve soluble proteins from Mtb, including one protein complex, were purified to homogeneity following recombinant expression in M. smegmatis. Protein purity was assessed both by size exclusion chromatography and mass spectrometry. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients. Conclusion and clinical relevance: The established workflow comprises a simple, cost-effective and scalable pipeline for production of soluble proteins from Mtb and can be used to prioritize immunogenic proteins suitable for use as diagnostic markers.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mycobacterium Tuberculosis H37rv

DISEASE(S): Tuberculosis

SUBMITTER: Joanna Kirkpatrick

LAB HEAD: Annabel Parret

PROVIDER: PXD004133 | Pride | 2018-06-27

REPOSITORIES: Pride

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Publications

A standardized production pipeline for high profile targets from Mycobacterium tuberculosis.

Milewski Morlin C MC Broger Tobias T Kirkpatrick Joanna J Filomena Angela A Komadina Dana D Schneiderhan-Marra Nicole N Wilmanns Matthias M Parret Annabel H A AH

Proteomics. Clinical applications 20160803 9-10

<h4>Purpose</h4>Tuberculosis is still a major threat to global health. New tools and strategies to produce disease-related proteins are quintessential for the development of novel vaccines and diagnostic markers.<h4>Experimental design</h4>To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. ...[more]

PMID: 27400835

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Project description:Introduction: Tuberculosis (TB) is a serious human disease caused by the bacterium Mycobacterium tuberculosis (Mtb). One third of the worldâs population is estimated to be latently infected with Mtb. Exact mechanisms and properties of latent TB infection (LTBI) are not fully elucidated. In essence, the pathogen can enter a dormant or latent state characterized by limited growth and metabolism, resulting in absence of clinical symptoms in the host, and most importantly by increased phenotypic tolerance to the commonly used medications, thereby allowing indefinite persistence in the human body. This persistence is the main reason why the current treatment of new-onset pulmonary TB is very long, consisting of a six months therapy of four antibiotics (rifampicin, isoniazid, pyrazinamide, and ethambutol for the first two months, and only rifampicin and isoniazid for the last four months). Current state of the art: To fight TB globally and more efficiently, it is essential to shorten the treatment for TB with new drugs that are efficient also against LTBI. To facilitate discovery of such drugs, in vitro models for LTBI can be used for high throughput screening of chemical libraries. Current in vitro models such as nutrient starvation (Betts et al. 2002), nutrient depletion (Hampshire et al. 2004; Rifat, Bishai, and Karakousis 2009), progressive hypoxia (Wayne and Hayes 1996), nitric oxide treatment (Martin I. Voskuil et al. 2003) and multiple stress (Deb et al. 2009) mimic the dormant state of Mtb, but the technical settings and equipment required for these models obstruct high throughput applications. Proposed model: The streptomycin (STR)-starved 18b model (SS18b) is a simple and drug discovery efficient Mtb latency model successfully applicable to both in vitro and in vivo settings (Zhang et al. 2012). It is based on the Mtb strain 18b, which is a STR-dependent mutant that enters a viable but nonreplicating state in the absence of STR (Hashimoto 1955). Despite the successful model, we know very little about 18b. How well does SS18b mimic the bacterial response to the complex host-pathogen interactions underlying LTBI, and how does the SS18b response compare to that of other dormancy models are still open questions. In this work we analyse the complete genome of 18b and describe the transcriptomic response of 18b to STR depletion. 12 samples were analyzed. Four biological replicates were sampled during the exponential growth phase, in presence of streptomycin. Four biological replicates derived after 2 weeks of streptomycin depletion, and two biological replicates derive after 4 weeks of streptomycin depletion. Two replicates derive from the resubmission of streptomycin after four weeks of depletion.

Dataset Information

Mycobacterium tuberculosis protein production pipeline

Publications

A standardized production pipeline for high profile targets from Mycobacterium tuberculosis.

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