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-P _cap -HC and pTSSCm-P _cap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (P _cap ) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His ( rgs - his ₆ ). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T ₁ , 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-P _cap -HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-P _cap ). The new constructs displayed increased plasmid yield and segregational stability in S. aureus . Furthermore, pBUS1-P _cap -HC and pTSSCm-P _cap offer the potential to generate C-terminal RGS-His ₆ translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs - his ₆ codons to the 3′ end of the target gene. The generation of the rgs - his ₆ codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm (44) inserted downstream of P _cap . The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.