Joanna S. Albala

From genes to proteins: high-throughput expression and purification of the human proteome.

The development of high‐throughput methods for gene discovery has paved the way for the design of new strategies for genome‐scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high‐throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high‐throughput expression system for protein production.

Array-based proteomics: the latest chip challenge

Array-based protein technologies are emerging for basic biological research, molecular diagnostics and therapeutic development with the potential of providing parallel functional analysis of hundreds or perhaps hundreds of thousands of proteins simultaneously. Array-based methods are becoming prevalent within proteomics research due to the desire to analyze proteins in an analogous format to that of the DNA microarray. Novel protein biochips are under development in academic laboratories and emerging biotechnology companies to advance the pace and scope of scientific discovery. This review will define array-based proteomics, its current applications and future directions, as well as examine the challenges and limitations of this projected billion dollar industry.

Accelerating code to function: sizing up the protein production line.

High-throughput biology has been pioneered by genomics through the application of robotics to expedite DNA-sequencing projects. Advances in high-throughput protein methods are needed to drive the protein production line for high-throughput structural and functional analysis of newly discovered genes. This will require the development and application of a variety of recombinant-protein expression systems to produce the diversity of proteins from both humans and model organisms.

Protein Expression Arrays for Proteomics

As biology approaches the 50th year of deciphering the DNA code, the next frontier toward understanding cell function has protein biochemistry in the form of structural and functional proteomics. To accomplish the needs of proteomics, novel strategies must be devised to examine the gene products or proteins, emerged as en masse. The authors have developed a high-throughput system for the expression and purification of eukaryotic proteins to provide the resources for structural studies and protein functional analysis. The long-term objective is to overexpress and purify thousands of proteins encoded by the human genome. This library of proteins--the human proteome--can be arrayed in addressable format in quantities and purities suitable for high-throughput studies. Critical technology involved in efficiently moving from genome to proteome includes parallel sample handling, robust expression, and rapid purification procedures. Automation of these processes is essential for the production of thousands of recombinant proteins and the reduction of human error.

Hitting the Spot: The Promise of Protein Microarrays

With the thrust of scientific endeavormoving from genomics to proteomics, the protein array provides a powerful means by which to examine hundreds to thousands of proteins in parallel. A result of the many genome projects has been the advance of automation and robotic procedures to manipulate biomolecules using a high-throughput, systematic approach. The promise of the protein microarray is the ability to interrogate a large number of proteins simultaneously in a high-density format for disease diagnosis, prognosis or efficacy of therapeutic regime as well as for biochemical analysis. Similar to aDNAmicroarray, each spot on a protein array can be identified based on its addressability on the planar surface.

Automated Construction of an Open Reading Frame Library from Sinorhizobium meliloti

An automated, high-throughput, open reading frame (ORF) library construction process has been developed. ORFs from genomic DNA of the microbe Sinorhizobium meliloti were amplified by PCR and cloned into the library vector by homologous recombination instead of traditional ligation. From 960 targets, we successfully generated 723 (75.3%) ORFs from the initial PCR. After cloning the successful samples into the library vector, transforming into E. coli and PCR colony screening, 371 (38.6% overall) ORFs were placed into the new library and sequenced. Our prototype library contained 314 (32.7% overall) clones with sequence identity to the Sinorhizobium meliloti genome.

Building biochips: a protein production pipeline

Protein arrays are emerging as a practical format in which to study proteins in high-throughput using many of the same techniques as that of the DNA microarray. The key advantage to array-based methods for protein study is the potential for parallel analysis of thousands of samples in an automated, high-throughput fashion. Building protein arrays capable of this analysis capacity requires a robust expression and purification system capable of generating hundreds to thousands of purified recombinant proteins. We have developed a method to utilize LLNL-I.M.A.G.E. cDNAs to generate recombinant protein libraries using a baculovirus-insect cell expression system. We have used this strategy to produce proteins for analysis of protein/DNA and protein/protein interactions using protein microarrays in order to understand the complex interactions of proteins involved in homologous recombination and DNA repair. Using protein array techniques, a novel interaction between the DNA repair protein, Rad51B, and histones has been identified.

Chips to Hits 2001: An Array of Parallel Technologies Converge

Chip users, vendors and other visionary researchers converged on sunny San Diego for the Chips to Hits meeting, October 28 – November 1, sponsored by IBC Conferences, Inc. This conference has become an industry standard not to be missed. Now in its eight year, the event showcases progress in the ever-expanding chip field – from academics to industry, siliconized glass slides to in silico data analysis. It has also become a launching pad for emerging technologies and rising biotech companies. The conference has grown in attendance every year and long-time attendee Mark Shena (TeleChem, Inc.) remarked on the recent and rapid expansion of the conference from its early years of about 60 scientists exchanging ideas and techniques at the Claremont Hotel in Berkeley. The program this year hosted over 1300 attendees and included three preconference sessions in surface chemistry for biochips, bioinformatics and nanobiotechnology as well as product demonstrations and technology workshops. General sessions were held in the areas of: • Commercialization of drug discovery technologies • Evolving roles, opportunities and challenges for chips • Emerging technologies • Protein microarrays • Microarrays for genomics • Microarrays for diagnostics The emphasis of this account will be focused on excerpts from presentations in the protein microarray and emerging technologies sessions. It is noteworthy that although this report reflects my personal bias towards proteomics, the conference in general seems to have veered from a strict DNA microarray course, being dominated by new technologies particularly geared toward understanding protein function. These sessions also reflect the broadening use of parallel technologies not confined to a planar surface, with multiplexing capabilities and emphasis on differential labeling and detection techniques.