PXD060310

PXD060310 is an original dataset announced via ProteomeXchange.

Title	pTripleTREP – A vector for tightly controlled expression and purification of virulence factors in Staphylococcus aureus
Description	Background: Recombinant proteins are an important building block towards understanding the virulence mechanisms and pathophysiology of the major human pathogen Staphylococcus aureus. Of particular interest are secreted virulence factors. However, due to their potential toxicity and specific post-translational processing, virulence factors are difficult targets for heterologous protein production. Purified proteins with native conformation and satisfying purity can therefore often only be achieved by elaborate multi-step purification workflows. Homologous expression in S. aureus theoretically offers a beneficial alternative in this regard. However, due to the lack of systems with both tightly controlled expression and subsequent efficient purification, it is rarely used. Results: To bridge this gap, we present pTripleTREP as a versatile expression vector for S. aureus, which enables the homologous expression and purification of staphylococcal virulence factors. Based on a strong SigA-dependent staphylococcal promoter overlapped by three tetracycline responsive elements (TRE), the target gene promoter ensures tight repression under control conditions but high expression levels after induction. This allowed very precisely controlled production of the exemplary targets, serine protease-like protein A (SplA) and B (SplB). A simple single-step protein purification workflow via Twin-Strep-tag and Strep-Tactin®XT coated magnetic beads resulted in endotoxin-free Spl samples with purities above 99 %. The homologous production host, thereby, facilitates native secretion and maturation without the need to engineer the target gene sequence. Correct cleavage of the signal peptide and the corresponding enzymatic activity of the generated protein products were demonstrated with SplA and B. Conclusion: The expression vector pTripleTREP adds an important element to the staphylococcal molecular toolbox, facilitating the tightly controlled homologous expression and native but rapid purification of secreted staphylococcal virulence factors. The optimised architecture and genetic features of the vector additionally provide a good basis for further applications such as plasmid-based complementation or interaction studies. Thus, pTripleTREP will support research on the role of staphylococcal virulence factors aiming at future therapies to combat this pathogen.
HostingRepository	PRIDE
AnnounceDate	2025-05-26
AnnouncementXML	Submission_2025-05-25_16:07:04.179.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hannes Wolfgramm
SpeciesList	scientific name: Staphylococcus aureus; NCBI TaxID: 1280;
ModificationList	monohydroxylated residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2025-01-29 01:31:11	ID requested
⏵ 1	2025-05-25 16:07:05	announced

10.1186/s12934-025-02736-7;

Wolfgramm H, Saade C, Harms M, Busch LM, Lange J, Schedlowski M, Surmann K, Gesell Salazar M, Hentschker C, Steil L, Michalik S, V, ö, lker U, Reder A, pTripleTREP - A vector for tightly controlled expression and purification of virulence factors in Staphylococcus aureus. Microb Cell Fact, 24(1):115(2025) [pubmed]

submitter keyword: Purity, Recombinant protein, Signal peptide,Staphylococcus aureus

Uwe Völker
contact affiliation	Interfaculty Institute of Genetics and Functional Genomics, Department of Functional Genomics, University Medicine Greifswald, Greifswald, Germany
contact email	voelker@uni-greifswald.de
lab head
Hannes Wolfgramm
contact affiliation	University Medicine Greifswald
contact email	Hannes.wolfgramm@uni-greifswald.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD060310
     1. Label: PRIDE project
     2. Name: pTripleTREP – A vector for tightly controlled expression and purification of virulence factors in Staphylococcus aureus