Protein production from the structural genomics perspective: achievements and future needs

Abstract

Despite a multitude of recent technical breakthroughs speeding high-resolution structural analysis of biological macromolecules, production of sufficient quantities of well-behaved, active protein continues to represent the rate-limiting step in many structure determination efforts. These challenges are only amplified when considered in the context of ongoing structural genomics efforts, which are now contending with multi-domain eukaryotic proteins, secreted proteins, and ever-larger macromolecular assemblies. Exciting new developments in eukaryotic expression platforms, including insect and mammalian-based systems, promise enhanced opportunities for structural approaches to some of the most important biological problems. Development and implementation of automated eukaryotic expression techniques promises to significantly improve production of materials for structural, functional, and biomedical research applications.

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative

“…Thus, it is unlikely that determining structures of bacterial proteins (i.e., homologs) will significantly bolster the number of computationally modelable structures in the proteomes of eukaryotic organisms. Adequate coverage of eukaryotic genomes will require direct targeting of these proteins, which will necessitate enhanced investment in eukaryotic expression systems (24).…”

The MultiBac Baculovirus/Insect Cell Expression Vector System for Producing Complex Protein Biologics

“…A very large number of proteins, their mutants and variants have been produced recombinantly, and their structure and function determined at high resolution. The availability of entire genomes has made it possible to address the protein repertoire of cells on a systems-wide scale, applying high-throughput technologies [35]. Recombinant protein expression in E.coli as a prokaryotic expression host has become commonplace in molecular biology laboratories world-wide.…”

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

“…Efficient DNA assembly methods to generate heterologous expression constructs have been implemented in concerted 'omics' efforts to analyze proteins system-wide, in highthroughput. Structural genomics consortia were established to determine atomic structures, seemingly in an industrial mode [27,28]. Automation and robotics have become a prerogative; as a consequence, traditional cloning methodologies were progressively replaced by more advanced methods [1,[29][30][31][32].…”

“…The underlying technologies were perfected over the years to a level that they could be productively harnessed in ambitious, highly parallelized 'omics' programs aimed at genome-and proteome wide studies of proteins in many organisms including humans [28]. In these research undertakings, protein encoding genes are synthesized, manipulated, varied and delivered into recombinant expression hosts on an industrial scale to enable high-throughput structure determination, populating protein structure databases such as the protein data bank (PDB) with unmatched efficiency and breathtaking speed, ushering in a new age of protein structural and functional research.…”