Solution Structure of the Immunodominant Domain of Protective Antigen GNA1870 of Neisseria meningitidis

GNA1870, a 28-kDa surface-exposed lipoprotein of Neisseria meningitidis recently discovered by reverse vaccinology, is one of the most potent antigens of Meningococcus and a promising candidate for a universal vaccine against a devastating disease. Previous studies of epitope mapping and genetic characterization identified residues critical for bactericidal response within the C-terminal domain of the molecule. To elucidate the conformation of protective epitopes, we used NMR spectroscopy to obtain the solution structure of the immunodominant 18-kDa C-terminal portion of GNA1870. The structure consists of an eight-stranded antiparallel beta-barrel overlaid by a short alpha-helix with an unstructured N-terminal end. Residues previously shown to be important for antibody recognition were mapped on loops facing the same ridge of the molecule. The sequence similarity of GNA1870 with members of the bacterial transferrin receptor family allows one to predict the folding of this class of well known bacterial antigens, providing the basis for the rational engineering of high affinity B cell epitopes

The new pLAI (<it>lux </it>regulon based auto-inducible) expression system for recombinant protein production in <it>Escherichia coli</it>

<p>Abstract</p> <p>Background</p> <p>After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction) prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in <it>E. coli</it>.</p> <p>Result</p> <p>The newly designed pLAI expression system places the expression of recombinant proteins in <it>Escherichia coli </it>under control of the regulatory genes of the <it>lux </it>regulon of <it>Vibrio fischeri</it>'s Quorum Sensing (QS) system.</p> <p>The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of <it>V. fischeri </it>for expression in <it>E. coli </it>upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression.</p> <p>Conclusion</p> <p>Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene independently from external inducers or drastic changes in metabolic conditions and enabling tight regulation of expression.</p

A quantitative portrait of three xylanase inhibiting protein families in different wheat cultivars using 2D-DIGE and multivariate statistical tools

Wheat grains contain three classes of xylanase inhibitors (XIs), i.e. TAXI (Triticum aestivum xylanase inhibitor), XIP (xylanase inhibiting protein) and TLXI (thaumatin-like xylanase inhibitor). These proteins are involved in plant defence and strongly affect cereal-based processes in which inhibitor-sensitive xylanases are used. This paper reports on the successful use of 2D-DIGE and tandem MS to discriminate XI (iso)forms and measures their qualitative and quantitative variation in six different wheat cultivars. In total, 18 TAXI-, 27 XIP- and 3 TLXI-type XI spots were identified. The multiple members of the large TAXI-gene family make a considerable contribution to the total TAXI population. For XIP-type XIs, XIP-I is expressed as the predominant form, albeit under variable degrees of PTMs. Only one TLXI genetic variant was identified, showing different degrees of glycosylation. Multiple comparison analysis revealed up to 5-fold intercultivar differences in protein level of XI (iso)forms. Evaluation of abundance patterns using multivariate statistical tools revealed highly distinctive as well as correlated levels of different XI forms among the six cultivars. As the constitutive (and induced) levels of the different XI (iso)forms, which are differentially regulated in response to various forms of stress in other wheat plant parts, considerably vary between the cultivars, it can be assumed that their degree of resistance against pathogenic attack differs. Similarities in abundance profiles between XI (iso)forms and pathogenesis-related chitinases are also in line with a role in plant defence.status: publishe