ABSTRACT
The secretion of recombinant proteins plays an important role in their economic production and purification. The secretion efficiency depends on the responsible signal peptide (SP) in combination with the target protein and the given host and cannot be predicted so far. Due to its high plasmid stability, the lack of alkaline extracellular proteases and only few contaminating extracellular host proteins, Priestia megaterium provides a promising alternative to common Bacillus species. For the development of an easy and fast cloning and screening system to identify the SP best suited to a distinct protein, a plasmid-based SP library containing all predicted 182 Sec-dependent SPs from P. megaterium was established. The splitting of the SPs into 10 groups of individual multi-SP plasmids (pMSPs) allows their grouped amplification and application in screening approaches. The functionality of the whole library was demonstrated by enhancing the amount of the already well-secreted α-amylase AmyE by 1.6-fold. The secretion of a novel penicillin G acylase, which remained as insoluble protein inside the cells, as its native SP is unsuitable for secretion in P. megaterium, could be enhanced even up to 29-fold. Overall, only around 170 recombinant P. megaterium clones based on 50 inserted SPs had to be screened to achieve sufficient amounts for further enzyme characterizations. Thus, this newly developed plasmid-based genetic tool applicable for P. megaterium and also other Bacillus species facilitates the identification of suitable SPs for secretion of recombinant proteins.
ABSTRACT
We investigate the immunoreactivity of serine/threonine kinase 33 (Stk33) and of vimentin in the brain of mouse, rat and hamster. Using a Stk33-specific polyclonal antibody, we show by immunofluorescence staining that Stk33 is present in a variety of brain regions. We found a strong staining in the ependymal lining of all cerebral ventricles and the central canal of the spinal cord as well as in hypothalamic tanycytes. Stk33 immunoreactivity was also found in circumventricular organs such as the area postrema, subfornical organ and pituitary and pineal glands. Double-immunostaining experiments with antibodies against Stk33 and vimentin showed a striking colocalization of Stk33 and vimentin. As shown previously, Stk33 phosphorylates recombinant vimentin in vitro. Co-immunoprecipitation experiments and co-sedimentation assays indicate that Stk33 and vimentin are associated in vivo and that this association does not depend on further interacting partners (Brauksiepe et al. in BMC Biochem 9:25, 2008). This indicates that Stk33 is involved in the dynamics of vimentin polymerization/depolymerization. Since in tanycytes the vimentin expression is regulated by the photoperiod (Kameda et al. in Cell Tissue Res 314:251-262, 2003), we determine whether this also holds true for Stk33. We study hypothalamic sections from adult Djungarian hamsters (Phodopus sungorus) held under either long photoperiods (L:D 16:8 h) or short photoperiods (L:D 8:16 h) for 2 months. In addition, we examine whether age-dependent changes in Stk33 protein content exist. Our results show that Stk33 in tanycytes is regulated by the photoperiod as is the case for vimentin. Stk33 may participate in photoperiodic regulation of the endocrine system.
