High-throughput production of prokaryotic membrane proteins

Dobrovetsky, E.; Lu, Ming; Andorn-Broza, Ronit; Khutoreskaya, G.; Bray, James E.; Savchenko, Alexei; Arrowsmith, C.H.; Edwards, A.M.; Koth, Christopher M.

doi:10.1007/s10969-005-1363-5

Cited by 45 publications

(39 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This protein-detergent complex (PDC) is often heterogeneous, creating numerous problems in purification and crystallization. Optimizing purification, assaying protein function, and crystallization all require milligram quantities of protein, and MP expression is therefore a limiting step in macromolecular structure determination (Dobrovetsky et al 2005;Eshaghi et al 2005;Korepanova et al 2005;Columbus et al 2006;Surade et al 2006). One recognized alternative is cell-free (CF) expression (Klammt et al 2004).…”

Section: Introductionmentioning

confidence: 99%

Cell‐free complements in vivo expression of the E. coli membrane proteome

et al. 2007

View full text Add to dashboard Cite

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cellfree expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.Keywords: cell-free protein expression; integral membrane proteins; structural genomics; high-throughput protein expression Integral membrane proteins (MPs), despite their biological importance, currently account for <1% of all known high resolution protein structures. MPs are notoriously difficult to work with, and expression, detergent solubilization, purification, and crystallization all present unique challenges over their soluble counterparts (White 2004). MPs generally express at much lower levels than soluble proteins and, when in vivo overexpression is successful, the protein can be cytotoxic or incorporated into insoluble inclusion bodies. Following successful MP expression, a suitable detergent condition must also be found that simultaneously extracts the protein from the membrane while retaining the native fold and function. This protein-detergent complex (PDC) is often heterogeneous, creating numerous problems in purification and crystallization. Optimizing purification, assaying protein function, and crystallization all require milligram quantities of protein, and MP expression is therefore a limiting step in macromolecular structure determination (Dobrovetsky et al. 2005;Eshaghi et al. 2005;Korepanova et al. 2005;Columbus et al. 2006;Surade et al. 2006). One recognized alternative is cell-free (CF) expression (Klammt et al. 2004). 4 These authors contributed equally to this work.

show abstract

Section: Introductionmentioning

confidence: 99%

Cell‐free complements in vivo expression of the E. coli membrane proteome

et al. 2007

View full text Add to dashboard Cite

show abstract

“…To meet this demand, many large projects are devoted to developing methods to generate large numbers of purified proteins. However, the task is proving challenging: on average, for proteins from prokaryotes, only 50-70% of soluble proteins and 30% of membrane proteins can be readily expressed in recombinant form, and only 30-50% of these expressed proteins can be purified to homogeneity (1,2). The success rates for human proteins are predicted to be significantly lower.…”

mentioning

confidence: 99%

Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination

Vedadi

Niesen

Allali-Hassani

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

533

526

View full text Add to dashboard Cite

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.chemical biology ͉ crystallography ͉ human S tructural, functional, and chemical genomics (proteomics) are disciplines that aim to determine the biochemical, cellular, and physiological functions of proteins on a genome scale. Many of the central, important experimental approaches that are involved, such as protein-based screens for small-molecule inhibitors, depend on the availability of purified and active proteins. To meet this demand, many large projects are devoted to developing methods to generate large numbers of purified proteins. However, the task is proving challenging: on average, for proteins from prokaryotes, only 50-70% of soluble proteins and 30% of membrane proteins can be readily expressed in recombinant form, and only 30-50% of these expressed proteins can be purified to homogeneity (1, 2). The success rates for human proteins are predicted to be significantly lower.To improve the general rates of protein purification, efforts have focused largely on alterations of the recombinant host, the expression conditions, changes of the construct encoding the protein, and the purification conditions. It is also known that the expression and purification of a protein can be improved significantly by the addition of a specific ligand, which serves to stabilize the protein, thereby reducing its propensity to unfold, aggregate, or succumb to proteolysis. This parameter has not been studied systematically, although in individual cases the addition of a specific ligand has had dramatic effects. For example, the recombinant expression of the guinea pig and human forms of the enzyme 11␤-hydroxysteroid dehydrogenase-1 in bacteria was increased dramatically by the addition of an inhibitor of the enzyme to the growing cells (3) Wu, K. L. Kav...

show abstract

“…However, the task has proved challenging: on average, for proteins from bacteria, only 50-70% of soluble proteins and 30% of membrane proteins can be readily expressed in recombinant form, and only 30-50% of these expressed proteins can be purified to homogeneity [48,49]. The coding regions of the P. falciparum genome contain 76.5%…”

Section: Discussionmentioning

confidence: 99%