Expression screening of integral membrane proteins from Helicobacter pylori 26695.
ABSTRACT The efficiency of Helicobacter pylori as a mucosal pathogen is caused by unique soluble and integral membrane proteins, which allow its survival at acidic pH and successful colonization of the gastric environment. With about one-fourth of the H. pylori's proteome comprising integral membrane proteins, the need for solution of their three-dimensional (3D) structures becomes persistent as it can potentially drive the generation of more effective drugs. This study presents a medium-throughput approach for cloning and expression screening of integral membrane proteins from H. pylori (26695) using Escherichia coli as the expression host. One-hundred sixteen H. pylori targets were cloned into two different vector systems and heterologously expressed in E. coli. Eighty-four percent of these proteins displayed medium to high expression. No clear-cut correlation was found between expression levels and number of putative transmembrane spans, predicted functionality, and molecular mass. Nonetheless, expression of transporters and hypothetical proteins < or =40 kDa with two to four transmembrane spans displayed generally high expression levels. To statistically strengthen the quality of the data from the medium-throughput approach, a comparison with data derived from robotic-based methodologies was conducted. Optimization of expression and solubilization conditions for selected targets was also performed. Seventeen targets have been purified and subjected to crystallization so far. Eighteen percent of these targets (2/17) produced crystals under specific sets of crystallization conditions.
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ABSTRACT: A century ago, secretions from the pancreas were described as 'hormones', which we now know are secreted from all ductless glands. The development of various technologies has already contributed a great deal -- and will undoubtedly offer more -- to our understanding of their mode of action.Nature Reviews Molecular Cell Biology 10/2002; 3(9):702-10. · 39.12 Impact Factor
Article: A flexible and economical medium-throughput strategy for protein production and crystallization.[show abstract] [hide abstract]
ABSTRACT: Large-scale structural genomics centres rely heavily on robotics to ensure that maximum throughput is achieved. However, the size and cost of these approaches is out of the reach of most academic structural biology efforts. A major challenge for such groups is to adapt current high-throughput schemes to a reasonable scale with the resources available. A flexible medium-throughput approach has been developed that is suitable for typical academic research groups. Following nested PCR, targets are routinely cloned into two Gateway expression vectors (pDEST15 for an N-terminal GST tag and pDEST17 for an N-terminal His tag). Expression of soluble recombinant protein in Escherichia coli is rapidly assessed in 96-well format. An eight-probe sonicator is utilized and a six-buffer lysis screen was incorporated to enhance solubility. Robotics is reserved for crystallization, since this is the key bottleneck for crystallography. Screening proteins with a 480-condition protocol using a Cartesian nanolitre-dispensing robot has increased crystallization success markedly, with an overall success rate (structures solved out of proteins screened) of 19%. The methods are robust and economical -- with the exception of the crystallization robot, investment in additional equipment has been minimal at 9000 US dollars. All protocols are designed for individuals so that graduate students and postdoctoral fellows gain expertise in every aspect of the structural pipeline, from cloning to crystallization.Acta Crystallographica Section D Biological Crystallography 11/2005; 61(Pt 10):1378-85. · 12.62 Impact Factor