SmpB, a small tmRNA binding protein, is essential for trans-translation. 6His and FLAG tagged SmpB was cloned from Mycobacterium tuberculosis H37Rv. It was expressed in Escherichia coli using the T7 promoter-polymerase system. Anti-FLAG M2 agarose was used for its purification. Mycobacterial SmpB copurifies with other proteins. We identified elongation factor EF-Tu in the purified SmpB preparations.
Mycobacterium smegmatis is a species of rapidly growing saprophytes with a number of properties that make it an effective vaccine vector. Recombinant M. smegmatis expressing protective antigens of different pathogens and molecules modulating the immune responses offers some potential for reduction of the burden of tuberculosis, HIV and hepatitis B infections. This paper discusses the molecular methods used to generate recombinant M. smegmatis and the results obtained with some of these recombinants.
Rv0802c acetyltransferase is a mycobacterial RNase E-associated protein. 6His and FLAG-tagged acetyltransferase was cloned from Mycobacterium tuberculosis H37Rv, expressed in Escherichia coli and partially purified. It is a 25 kDa protein showing a modest sequence homology with other acetyltransferases. The R-X-X-G-X-G sequence for acetyl-coenzyme A recognition and binding can be found in the molecule.
Cytokine production has been implicated in the pathogenic mechanisms of infections caused by the staphylococci, since these bacteria may act as strong cytokine inducers. To gain deeper insight into the Th1 immune response activated by these bacteria, we have analyzed the interferon (IFN), interleukin-12 (IL-12) and IL-18-inducing activities of different Staphylococcus aureus (S. aureus), S. epidermidis and S. saprophyticus strains in human monocytes and murine bone marrow macrophages. A large majority of the S. aureus strains elicited the simultaneous production of IL-12 p70 and IFN-a in the human monocytes, while the S. epidermidis and S. saprophyticus strains induced only a low level of production, if any, of these cytokines. Furthermore, a majority of the S. aureus strains induced significantly higher IL-12 p70 and IL-18 titers in the murine bone marrow macrophages than did the S. epidermidis and S. saprophyticus strains. As IL-12, IL-18 and IFN-a stimulate Th1 differentiation synergistically, we suggest that S. aureus strains bias the immune response toward a Th1 phenotype, whereas S. epidermidis and S. saprophyticus strains provide a weaker stimulus for the production of Th1-inducing cytokines, and accordingly possibly elicit a less extensive Th1-associated adaptive immunity.
possesses a type III secretion system (TTSS), which allows the bacteria to secrete effector molecules into the inclusion membrane and into the cytosol of the host cell. Low calcium response protein H (LcrH), as a part of the TTSS, is a chaperone protein expressed from the middle to late stages of the chlamydial developmental cycle. Gene of LcrH (CPn0811) in a 6His-tagged form was cloned from
CWL029, expressed and purified from
using the HIS-select TALON CellThru Resin. The purity was checked with mass spectrometry. The samples were used for immunization of BALB/c mice. The inducible
clone, which over-expresses the chlamydial LcrH, permits the study of the biological properties of this protein.