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Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells. [ABSTRACT FROM AUTHOR]

The human general transcription factor TFIIH is involved in both transcription and DNA repair. We have identified a structural domain in the core subunit of TFIIH, p62, which is absolutely required for DNA repair activity through the nucleotide excision repair pathway. Using coimmunoprecipitation experiments, we showed that this activity involves the interaction between the N-terminal domain of p62 and the 3' endonuclease XPG, a major component of the nucleotide excision repair machinery. Furthermore, we reconstituted a functional TFIIH particle with a mutant of p62 lacking the N-terminal domain, showing that this domain is not required for assembly of the TFIIH complex and basal transcription. We solved its three-dimensional structure and found an unpredicted pleckstrin homology and phosphotyrosine binding (PH/PTB) domain, uncovering a new class of activity for this fold. [ABSTRACT FROM AUTHOR]

Examines the existence of a eukaryotic XPB/ERCC3-like helicase in Mycobacterium leprae. Use of the characteristic patterns from eukaryotic XPB/ERCC3 homologues in searching the nucleotide database; Existence of a XPB/ERCC3-like proteins in a prokaryote.

Abstract: Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes. [Copyright &y& Elsevier]

Abstract: Purified protein expression level and quality are contingent upon specific host expression systems. This differential production is particularly observed for proteins of high molecular weight, hampering further structural studies. We developed an expression method aimed at producing proteins in Escherichia coli, insect, and mammalian systems. Our novel protocol was used to produce in large scale the full-length 160-kDa steroid receptor coactivator 1 (SRC-1), a coregulator of nuclear receptors. The results indicate that we can produce biologically active human SRC-1 in mammalian and insect cells in large scale. [Copyright &y& Elsevier]