Laboratory scale structural genomics.pdf

Laboratory scale structural genomics Brent W. Segelke1, Johana Schafer1, Matthew A. Coleman2, Tim P. Lekin1, Dominique Toppani1, Krzysztof J. Skowronek1, Katherine A. Kantardjieff3 Bernhard Rupp1,* 1Macromolecular Crystallography and Structural Genomics Group, Biology and Biotechnology Research Program, P.O. Box 808, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA; 2Health Effects Genetics Division, Biology and Biotechnology Research Program, P.O. Box 808, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA; 3W.M. Keck Foundation Center for Molecular Structure, Department of Chemistry and Biochemistry, California State University Fullerton, 800 N. State College Blvd., Fullerton, CA 92834-6866, USA; *Author for correspondence (tel: +1-925-423-3273; fax: +1-925-424-3130; e-mail: br@) Received 13 June 2003; accepted in revised form 22 October 2003 Key words: cloning, crystallization, expression, high throughput, TB structural genomics Abstract At Lawrence Livermore National Laboratory, the development of the TB structural genomics consortium crys- tallization facility has paralleled several local proteomics research efforts that have grown out of gene expression microarray and comparative genomics studies. Collective experience gathered from TB consortium labs and other centers involved in the NIH-NIGMS protein structure initiative allows us to explore the possibilities and chal- lenges of pursuing structural genomics on an academic laboratory scale. We discuss our procedures and proto- cols for genomic targeting approaches, primer design, cloning, small scale expression screening, scale-up and purification, through to automated crystallization screening and data collection. The procedures are carried out by a small group using a combination of traditional approaches, innovative molecular biochemistry approaches, software automation, and a modest investment in robotic equipment. Introduction The success of the genome sequencing projects d