New shuttle vector-based expression system to generate polyhistidine-tagged fusion proteins in Staphylococcus aureus and Escherichia coli

Applied and Environmental Microbiology
Sybille Schwendener, Vincent Perreten

Abstract

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by re...Continue Reading

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Aug 6, 2014·Antimicrobial Agents and Chemotherapy·Juliette R K WipfVincent Perreten

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Citations

Feb 22, 2017·Journal of the American Chemical Society·Weizhong ChenQuanjiang Ji
Apr 26, 2017·Antimicrobial Agents and Chemotherapy·Juliette R K WipfVincent Perreten
May 13, 2017·MSphere·Sabrina N AndreisSybille Schwendener
Oct 30, 2020·The Journal of Antimicrobial Chemotherapy·Javier Eduardo FernandezSybille Schwendener
Aug 24, 2017·International Journal of Biological Macromolecules·Jashandeep KaurJagdeep Kaur

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