New shuttle vector-based expression system to generate polyhistidine-tagged fusion proteins in Staphylococcus aureus and Escherichia coli.
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 religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 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 Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.
Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Sybille
S
Schwendener
Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland vincent.perreten@vetsuisse.unibe.ch.
Vincent
V
Perreten
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D008780:Methyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018741:Replication Origin; D012093:Replicon; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000235:genetics*
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase