The aims of high-throughput (HTP) protein production systems are to obtain well-expressed and highly soluble proteins, which are preferred candidates for use in structure-function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96-well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well-expressed soluble fusion protein, total cell lysates of bacteria culture (∼1.5 mL) were subjected to high-speed centrifugation in a 96-tube format and analyzed by multiwell denaturing SDS-PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis. . Cloning and expression in Escherichia coli are favored in many instances because E. coli has relatively simple genetics, is well characterized, has a relatively rapid growth rate, and has few post-translational protein modifications. One disadvantage, however, of expressing heterologous proteins in E. coli is that proteins are frequently expressed as insoluble aggregated folding intermediates, known as inclusion bodies (Paul et al. 1983). Although it may be possible to increase protein solubility by optimizing expression condition or by refolding the recombinant proteins, in the interests of throughput, only a single set of growth or folding conditions can be used.Gene fusion is another approach that has been successfully used for producing soluble heterologous proteins in E. coli (Uhl'en and Moks 1990). Several carrier proteins are widely used in gene fusion, including thioredoxin (Trx), maltose-binding protein (MBP), glutathione S-transferase (GST), intein, calmodulin-binding protein (CBP), NusA, and cellulose-associated protein (CAP). Although the use of these carrier proteins has resulted in the successful overexpression of many heterologous proteins, each was tested Reprint requests to: Ting-Fang Wang, Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, Republic of China; e-mail: tfwang@ gate.sinica.edu.tw; fax: 886-2-27889759. 1These two authors contributed equally to this work.

Overproduction of proteins from cloned genes using fusion protein expression vectors in Escherichia coli and eukaryotic cells has increased the quantity of protein produced. This approach has been widely used in producing soluble recombinant proteins for structural and functional analysis. One major disadvantage, however, of applying this approach for clinical or bioindustrial uses is that proteolytic removal of the fusion carrier is tedious, expensive, and often results in products with additional amino acid residues than the native proteins. Here we describe a new method for productions of native proteins with original amino termini in vivo via intracellular self-cleavage of the fusion protein using tobacco etch virus (TEV) protease. Our design allows one to simultaneously clone any gene into multiple fusion protein vectors using two unique cloning sites (i.e., SnaBI and XhoI) without restriction digestion, and then rapidly identifies those constructs producing soluble native proteins. This method will make the fusion protein approach more feasible for protein drug research.Keywords: tobacco etch virus protease; sticky-end PCR; fusion protein approach The fusion protein approach has been widely applied in modern biology and protein science research. More than 20 carrier proteins or affinity tags are available now for this approach to produce soluble heterologous proteins in various host organisms (Sambrook and Russell 2000). Although the use of these carrier proteins has resulted in successful overexpression of many heterologous proteins, each must be tested empirically, and certainly may not possess maximal solubility. Moreover, each expression scenario requires a specific vector. Recloning of the passenger protein gene into each of specific vectors is extremely labor-intensive. Recombinational cloning methods (Liu et al. 1998;Hartley et al. 2000) and sticky-end PCR cloning strategy (Shih et al. 2002;Wang and Wang 2004) were applied to minimize efforts required for alternate expression, and also allowed one to develop a high-throughput system to screen for soluble recombinant proteins.Because of concerns about the impact of carrier proteins or affinity tags on the structure or activity of a passenger target protein, it is ordinarily desirable to remove them. Typically, passenger target protein is separated from the fusion carrier by site-specifically proteolysis after affinity chromatography. It is this step that was considered to be the Achilles' heel of fusion protein approach, particularly in applications such as structural biology or protein-drug production. It is relatively common to encounter a situation in which fusion carriers cannot be processed effectively because of the steric hindrance at the cleavage site. Tedious

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Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.