Amit Noheria - Academia.edu (original) (raw)

Papers by Amit Noheria

Structure, 2004

011 have "Expression_System" records (http:// Bronx, New York 10461 www.rcsb.org). Of these, over... more 011 have "Expression_System" records (http:// Bronx, New York 10461 www.rcsb.org). Of these, over 90% were produced using 3 Diabetes Research and Training Center and E. coli; ‫%5.3ف‬ were produced in yeast; ‫%5.2ف‬ with Department of Medicine insect cells; and ‫%5.1ف‬ using mammalian cells. The Division of Endocrinology remaining ‫%3ف‬ of these structures were determined Albert Einstein College of Medicine using proteins expressed in other systems, including 1300 Morris Park Avenue bacterial hosts such as B. subtilis and cell-free expres-Bronx, New York 10461 sion systems. The success of bacteria-based expres-4 Howard Hughes Medical Institute sion systems arises from several factors, including the Columbia University ease with which such organisms can be genetically ma-New York, New York 10032 nipulated; the thorough understanding of their transcrip-5 Naiomi Berrie Diabetes Center tion and translation machinery, which has led to the Columbia University ability to achieve high levels of protein expression; the New York, New York 10032 rapidity of their growth; and the relatively low cost of 6 Edward S. Harkness Eye Institute their use. Despite these advantages, bacterial systems Columbia University often fail in their application to the expression of eukary-New York, New York 10032 otic proteins (Baneyx, 1999; Makrides, 1999; Geisse et al. , 1996). Failure to achieve acceptable expression often arises from toxicity of the foreign protein or its inabil-Summary ity to fold or be targeted properly in the bacterial cell. Such problems inevitably result in low levels of expres-The expression of mammalian proteins in sufficient sion or protein misfolding (Baneyx, 1999; Makrides, abundance and quality for structural studies often pre-1999; Geisse et al., 1996). Thus, despite drawbacks in sents formidable challenges. Many express poorly in efficiency, alternative expression systems based on bacterial systems, whereas it can be time consuming eukaryotic hosts have been developed for large-scale and expensive to produce them from cells of higher protein production. These include expression in yeast, organisms. Here we describe a procedure for the diinsect cells, and mammalian cells, all of which have been rect selection of stable mammalian cell lines that exused successfully in producing proteins for structure press proteins of interest in high yield. Coexpression determination. of a marker protein, such as green fluorescent protein, Expression of mammalian proteins has proven to be is linked to that of the desired protein through an interparticularly challenging in bacterial systems. The sucnal ribosome entry site in the vector that is transfected cess rate has been better in heterologous eukaryotic into cells in culture. The coexpressed marker is used cell systems, such as yeast, baculovirus-infected insect to select for highly expressing clonal cell lines. Applicells, and stably transformed insect cells, but optimal cations are described to a membrane protein, the expression of some mammalian proteins may require 5HT2c serotonin receptor, and to a secreted cysteinemammalian host cells. These proteins evolved in a rich protein, resistin. Besides providing an expeditious mammalian cellular milieu, and it is understandable that means for producing mammalian proteins for strucboth proper folding and stability may depend on this tural work, the resulting cell lines also readily support environment. Unique properties of the mammalian cell tests of functional properties and structure-inspired environment that may facilitate homologous expression hypotheses.

Structure, 2004

011 have "Expression_System" records (http:// Bronx, New York 10461 www.rcsb.org). Of these, over... more 011 have "Expression_System" records (http:// Bronx, New York 10461 www.rcsb.org). Of these, over 90% were produced using 3 Diabetes Research and Training Center and E. coli; ‫%5.3ف‬ were produced in yeast; ‫%5.2ف‬ with Department of Medicine insect cells; and ‫%5.1ف‬ using mammalian cells. The Division of Endocrinology remaining ‫%3ف‬ of these structures were determined Albert Einstein College of Medicine using proteins expressed in other systems, including 1300 Morris Park Avenue bacterial hosts such as B. subtilis and cell-free expres-Bronx, New York 10461 sion systems. The success of bacteria-based expres-4 Howard Hughes Medical Institute sion systems arises from several factors, including the Columbia University ease with which such organisms can be genetically ma-New York, New York 10032 nipulated; the thorough understanding of their transcrip-5 Naiomi Berrie Diabetes Center tion and translation machinery, which has led to the Columbia University ability to achieve high levels of protein expression; the New York, New York 10032 rapidity of their growth; and the relatively low cost of 6 Edward S. Harkness Eye Institute their use. Despite these advantages, bacterial systems Columbia University often fail in their application to the expression of eukary-New York, New York 10032 otic proteins (Baneyx, 1999; Makrides, 1999; Geisse et al. , 1996). Failure to achieve acceptable expression often arises from toxicity of the foreign protein or its inabil-Summary ity to fold or be targeted properly in the bacterial cell. Such problems inevitably result in low levels of expres-The expression of mammalian proteins in sufficient sion or protein misfolding (Baneyx, 1999; Makrides, abundance and quality for structural studies often pre-1999; Geisse et al., 1996). Thus, despite drawbacks in sents formidable challenges. Many express poorly in efficiency, alternative expression systems based on bacterial systems, whereas it can be time consuming eukaryotic hosts have been developed for large-scale and expensive to produce them from cells of higher protein production. These include expression in yeast, organisms. Here we describe a procedure for the diinsect cells, and mammalian cells, all of which have been rect selection of stable mammalian cell lines that exused successfully in producing proteins for structure press proteins of interest in high yield. Coexpression determination. of a marker protein, such as green fluorescent protein, Expression of mammalian proteins has proven to be is linked to that of the desired protein through an interparticularly challenging in bacterial systems. The sucnal ribosome entry site in the vector that is transfected cess rate has been better in heterologous eukaryotic into cells in culture. The coexpressed marker is used cell systems, such as yeast, baculovirus-infected insect to select for highly expressing clonal cell lines. Applicells, and stably transformed insect cells, but optimal cations are described to a membrane protein, the expression of some mammalian proteins may require 5HT2c serotonin receptor, and to a secreted cysteinemammalian host cells. These proteins evolved in a rich protein, resistin. Besides providing an expeditious mammalian cellular milieu, and it is understandable that means for producing mammalian proteins for strucboth proper folding and stability may depend on this tural work, the resulting cell lines also readily support environment. Unique properties of the mammalian cell tests of functional properties and structure-inspired environment that may facilitate homologous expression hypotheses.