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Protein Production and Crystallization Workshop

Location: NIH
Start Date: 4/9/2003 8:00 AM
End Date: 4/11/2003 8:00 AM

The Protein Structure Initiative (PSI) aims to provide one or more representative structures from each of the several thousand protein-domain families found in living organisms. PSI is in its third year, and the nine PSI pilot research centers have identified a number of important bottlenecks that need to be addressed in efforts to meet the goals of the program.  For example, it is accepted that a major challenge to high-throughput determination of protein structure is the production of protein samples and crystals that are suitable for structural analysis.

To facilitate an effective exchange of developments and advancements pilot research centers, the National Institute of General Medical Sciences (NIGMS) organized annual workshops on gene cloning and protein expression and purification.  The first such meeting took place in March 2002 at the National Institutes of Health (NIH), in Bethesda, Maryland (see /News/Meetings/pages/ProteinProduction).  In a second workshop the scope of the meeting was expanded to include crystallization. The Protein Production and Crystallization Workshop was held at NIH, in Bethesda, in April 2003.
 
Protein production for structural genomics comprises the following processes:
  • Selection of target proteins
  • Cloning and expression of recombinant proteins
  • Cell-culture fermentation
  • Enrichment with selenium isotopes for X-ray crystallography or with stable isotopes for nuclear magnetic resonance (NMR)
  • Purification of proteins
  • Analytic characterization of proteins
  • Growth of X-ray–quality protein crystals
Within the organization of the PSI research centers the results of these experimental processes are organized into databases, the product reagents organized into reagent libraries, and the issues associated with distributing these reagents to the research community are addressed.  
 
The 2003 NIGMS Protein Purification and Crystallization Workshop was organized with the main goal of providing an effective platform for scientists to share and exchange ideas and data, to discuss progress and problems, and to address the most challenging bottlenecks. Another purpose of the workshop was to encourage contacts and collaborations among the participating groups. The meeting brought together representatives from all nine P50 center grants and two P01 program project grants and several recipients of NIH R01 and SBIR grants in the area of structural genomics methodology and technology development. Other attendees included representatives from similar international efforts in Canada, Europe, and Japan; NIGMS and other components of NIH; and other Federal agencies. A total of 110 participants met at this workshop. Nine invited speakers presented research results in areas relevant to the meeting topic, and representatives of the research centers and program projects described the bottlenecks in the research process and presented the study results. In addition, experts from the international community were invited to provide initial discussion points in all critical areas pertinent to the main topics of the workshop. Discussions focused on progress in high-throughput methods for cloning, production, purification, and crystallization of proteins for X-ray crystallography and NMR studies.
 
Workshop topics included the following:
  • Cloning technologies
  • Statistics on DNA sequence quality and error rates for polymerase chain reaction
  • Vectors and host strains
  • Complementary DNA (cDNA) clones or reagents
  • Technologies for eukaryotic expression
  • Approaches to solubilizing or refolding proteins or both
  • Issues related to metalloproteins and post-translational modification
  • High-density fermentation and scale-up of fermentation
  • Protein production and purification and parallel approaches to purification
  • Homogeneity and quality control of protein samples
  • Expression of membrane proteins
  • Reconstitution of proteins into membrane environments
  • Crystallization of membrane proteins
  • High-throughput crystallization
  • Crystallization optimization
  • Sample storage, reagent archiving, and record keeping
  • Robotic platforms for these procedures
  • Failures, problems, and bottlenecks
Scientists conducting research in all the PSI pilot projects and P01 pilot projects and investigators involved in several international efforts made presentations. For the first time, a poster session was included in the workshop. Several posters were selected for short oral communications, and an “open microphone” session was organized to provide for the reporting of new results. A session on databases, laboratory information management systems, and distribution of materials was also offered.  Abundant time for discussion was planned, and the workshop was completed with a summary discussion. The proceedings of the Protein Production and Crystallization Workshop will be published in the Journal of Functional and Structural Genomics (in early 2004). The organizing committee for the meeting has compiled information about research methods, technology, instrumentation, and contacts and has posted this resource on the PSI Web page (see /Initiatives/PSI/Meetings/pages/).
 
The workshop provided opportunity for updating the status of advancement in several important areas of the structural genomics pilot projects. In an effort to stimulate progress, researchers in these pilot projects have used a diversity of approaches, and it was clear from the presentations and the study results that this strategy is paying off.   First, the progress since last year in areas covered by the workshop is very impressive. Large-scale gene cloning and expression is ongoing in a number of centers, and thousands of genes have been cloned already. Protein production and crystallization are proceeding on a scale not observed previously. Second, several approaches are being developed, implemented, tested, and evaluated, yielding new observations and sometimes unexpected results. Moreover, numerous processes have been at least partly automated and made more efficient and cost-effective. Third, more challenging proteins are being examined in several centers, where scientists are studying cell free expression of eukaryotic proteins, expression and purification of membrane proteins, and refolding of proteins from inclusion bodies.  Fourth, large-scale evaluation of the results in the databases has begun to provide important correlations that may help to improve selection of target proteins and to optimize experiment design and methods.
 
These findings suggest that PSI pilot centers are already making important advances in the technology of protein production and crystallization for structural biology. The resulting technology is expected to have broad value to the biological science community.
 
PP&CW Organizing Committee:
 
Michael Adams, Ph.D.
University of Georgia
Southeast Structural Genomics Consortium
 
Rosalind Kim, Ph.D.
Lawrence Berkeley National Laboratory
Berkeley Structural Genomics Center
 
Andrez Joachimiak, Ph.D.
Argonne National Laboratories
Midwest Structural Genomics Consortium
 
Gaetano T. Montelione, Ph.D.
Rutgers University
and Robert Wood Johnson Medical School, UMDNJ
Northeast Structural Genomics Consortium
 
John Norvell, Ph.D.
Protein Structure Initiative
National Institute of General Medical Sciences
National Institutes of Health

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Workshop Video Archive

A video archive of the 2003 Protein Production and Crystallization Workshop is available on the NIH Videocast Web site for each of the workshop's three days:

(Note: The above links require free RealPlayer software and 220Kbps LAN or 56Kbps dial-up bandwidth.)




This page last reviewed on November 14, 2014