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

Abstract

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. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties.

Proteome-scale purification of human proteins from bacteria

“…As experience with HT protein purification increases, it is likely that future approaches will use a limited menu of expression and purification conditions that among them could succeed with nearly all proteins (18,22). As shown here, the use of recombinational cloning facilitates the rapid transfer of the cDNAs to any needed expression vector.…”

Microarrays to characterize protein interactions on a whole‐proteome scale

“…At Protometrix, we employ a massively parallel baculovirus-based expression platform that has been adapted and optimized for high-throughput expression of mammalian proteins; we generally achieve an 80% or higher rate of success in producing soluble human proteins of the expected M r using this system ( Fig. 2), which is about two-fold higher than previous reports [25]. It should be pointed out that this kind of parallel production of thousands of proteins is achievable with high efficiency and at a reasonable cost because of the relatively small amount of protein required for microarrays.…”

A Proteomic Primer for the Clinician

“…Many research groups and companies have contributed tremendous effort in developing high-throughput protein purification methods, and recombinant proteins have been purified from E. coli, yeast, insects, and humans (70)(71)(72)(73)(74). Leuking and coworkers cloned cDNAs from human fetal brain tissues as C-terminal Hisx6-tagged fusions (75).…”

“…However, because eukaryotic proteins expressed in prokaryotic systems are not post-translationally modified, Zhu and coworkers has developed a high-throughput protein purification method from the budding yeast (71). For the same reason, Albala and associates chose 72 unique human cDNA clones to create an array of recombinant baculoviruses, from which 42% of the clones produced soluble fusion proteins in a 96-well format (74). Functional protein chips like traditional assays performed in microtiter plates (76) are suitable for a wide variety of biochemical analyses.…”