Klebsiella pneumoniae OmpA, the 40-kDa major protein of the outer membrane, was cloned and expressed in Escherichia coli. The recombinant protein was produced intracellularly in E. coli as inclusion bodies. Fusion of a short peptide to the N-terminus of native P40 facilitated high-level expression of the recombinant protein. Purified recombinant P40 was analyzed to verify purity and structural integrity. The molecular mass of purified recombinant P40 determined by electrospray mass spectrometry was 37 061 Da, in agreement with the theoretical mass deduced from the DNA sequence. Specific proliferation of recombinant-P40-primed murine lymph node cells in response to recombinant P40 stimulation in vitro indicated the presence of a T-cell epitope on recombinant P40. The induction of high serum antibody titers to a synthetic peptide derived from the attachment protein G of the respiratory syncytial virus when chemically coupled to recombinant P40 indicated that the protein had potent carrier properties. OmpA [20, 21] have been demonstrated to mediate expression of gram-negative bacteria, present at about 10 5 copies/cell. It is believed to occur in a monomeric form in its native state. A of peptides on the surface of live enterobacterial vectors. OmpC, the outer-membrane protein complex of Neisseria meningitidis, typical feature of OmpA is that it can be modified by heat: the mobility of Escherichia coli OmpA on SDS/PAGE decreases has been shown to be effective in humans as a conjugate vaccine with Haemophilus influenzae, pneumococcal and meningococcal when it is heated in the presence of SDS [1]. OmpA is highly conserved among gram-negative bacteria and is thought to con-capsular polysaccharides [22Ϫ25]. Other clinically useful carrier proteins have been derived from bacteria: diphteria and tetanus sist of two domains. The N terminus, including amino acid residues 1Ϫ170, forms a membrane-spanning domain and crosses toxoids are both successfully used in conjugated H. influenzae vaccines to transform the capsular polysaccharide, which is a Tthe outer membrane eight times in antiparallel β-strands, leading to a typical amphiphilic β-barrel [2Ϫ4]. The C-terminal moiety independent antigen, into a T-dependent antigen [26]. KeywordsIn this paper we describe the expression and production in of the protein is thought to be periplasmic [5]. The protein seems to be multifunctional. In addition to non-physiological functions, E. coli and purification of Klebsiella pneumoniae OmpA [27].Using various analytical criteria, the purity and structural integsuch as serving as a receptor for phages and colicins [6], it serves as a mediator in F-factor-dependent conjugation [7]. It is rity of the protein were evaluated. We demonstrate the presence of at least one T-cell epitope on the molecule and its potential also required for the structural integrity of the outer membrane and the generation of normal cell shape [8]. The capacity to use as a carrier protein for conjugated peptides. The immunological carrier properties of the recombin...
The somatostatin receptor subtype sst2 mediates both activation of a tyrosine phosphatase activity and inhibition of cell proliferation induced by somatostatin analogues.
We have previously shown that somatostatin promotes the stimulation of a membrane tyrosine phosphatase activity in pancreatic cells. To gain insight into the mechanism of somatostatin action, we purified somatostatin-receptor complexes from somatostatin 28-prelabelled rat pancreatic plasma membranes by immunoaffinity chromatography using immobilized antibodies raised against the N-terminal part of somatostatin 28, somatostatin 28 (1-14), which is not involved in receptor-binding-site recognition. After SDS gel electrophoresis a band with a molecular mass of 87 kDa was identified in the affinity-purified material as the somatostatin receptor. The 87 kDa protein was not observed when the membrane receptors were solubilized in a free unoccupied or somatostatin 14-occupied form, or when nonimmune serum replaced the anti-[somatostatin 28 (1-14)] anti-serum. Somatostatin 14 inhibited the appearance of the 87 kDa protein in the same range of concentrations that inhibit radioligand binding on pancreatic membranes. After somatostatin 28 treatment of membranes, purified somatostatin receptor preparations exhibited an elevated tyrosine phosphatase activity that dephosphorylated phosphorylated epidermal growth factor receptor and poly(Glu,Tyr). This activity was related to the presence of somatostatin receptors in purified material. It was increased by dithiothreitol and inhibited by orthovanadate. In purified material containing somatostatin receptors, anti-[Src homology 2 domains (SH2)]-containing tyrosine phosphatase SHPTP1 polyclonal antibodies identified a protein of 66 kDa which was not detected in the absence of somatostatin receptor. Furthermore, the anti-SHPTP1 antibodies immunoprecipitated specific somatostatin receptors from somatostatin-prelabelled pancreatic membranes and from untreated membranes. These results indicate that a 66 kDa tyrosine phosphatase related to SHPTP1 co-purifies with the pancreatic somatostatin receptors, and suggest that this protein is associated with somatostatin receptors at the membrane level.
Whereas agonist-directed differential signaling at a single receptor subtype has become an accepted pharmacological concept, distinct behaviors by ligands that are assumed to be antagonists is less documented. The intrinsic activity and capacity of antagonism for a new series of imidazoline-derived adrenergic ligands analogous to dexefaroxan were investigated by measuring two distinct signaling pathways at the recombinant human ␣ 2 -Adrenoceptors (␣ 2 ARs) are cell surface G protein-coupled receptors that bind native catecholamines and couple preferentially to the G i/o family of G proteins (Limbird, 1988). They are widely expressed in both the central and peripheral nervous systems (Eason and Liggett, 1993;Handy et al., 1993;Tavares et al., 1996) and have been shown to participate in a broad spectrum of physiological functions, which include inhibition of neurotransmitter release, regulation of blood pressure (both centrally and within the vasculature), sedation, analgesia, regulation of insulin release and lipolysis, renal function, and multiple behavioral and cognitive functions (Small and Liggett, 2001). The cellular effects of ␣ 2 AR activation include inhibition of adenylate cyclase, activaArticle, publication date, and citation information can be found at
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