title: Small-scale expression of proteins in E. coli.
abstract: Proteins participate in virtually every cellular activity, and a knowledge of protein function is essential for an understanding of biological systems. However, protein diversity necessitates the application of an array of in vivo and in vitro approaches for characterization of the functional and biochemical properties of proteins. Methods that enable production of proteins for in vitro studies are critical for determination of the molecular, kinetic, and thermodynamic properties of these molecules. Ideally, proteins could be purified from the original source; however, the native host is often unsuitable for a number of reasons. Consequently, systems for heterologous protein production are commonly used to produce large amounts of protein. Heterologous expression hosts are chosen using a number of criteria, including genetic tractability, advantageous production or processing characteristics (secretion or posttranslational modifications), or economy of time and growth requirements. The subcloning process also provides an opportunity to introduce purification tags, epitope tags, fusions, truncations, and mutations into the coding sequence that may be useful in downstream purification or characterization applications. Bacterial systems for heterologous protein expression have advantages in ease of use, cost, short generation times, and scalability. These expression systems have been widely used by high-throughput protein production projects and often represent an initial experiment for any expression target. Escherichia coli has been studied for many years as a model bacterial organism and is one of the most popular hosts for heterologous protein expression (Terpe, 2006). Its protein production capabilities have been intensively studied, and the ease of genetic manipulation in this organism has led to the development of strains engineered exclusively for use in protein expression. These resources are widely available from commercial sources and public repositories. Despite these advantages, many targets are unsuitable for expression in E. coli, and attempts will not yield protein that can be utilized in downstream applications. A thorough understanding of the protein target, the requirements of the final application, and available tools are all essential for planning a successful expression experiment. This protocol is designed to optimize expression and solubility using an E. coli host and expression vector with an IPTG-inducible T7 promoter. The general features of the method are easily extended to other organisms and expression systems. Small-scale expression cultures are used to identify the optimum expression parameters for a given target. Thorough analysis of the total cell content and soluble fraction is used to screen out failed targets and those unlikely to succeed in large-scale purification cultures. The protocol listed here can be used in individual tubes for a small number of targets or adapted for use in 48-well plates for high throughput applications (Abdullah et al., 2009). Using the same culture for initial expression analysis and solubility analysis reduces variability between expression trials and saves the time required to produce separate cultures.
authors.affiliation: Biosciences Division, Argonne National Laboratory, Lemont, IL, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Zerbs
authors.affiliation: Biosciences Division, Argonne National Laboratory, Lemont, IL, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Giuliani
authors.affiliation: Biosciences Division, Argonne National Laboratory, Lemont, IL, USA. Electronic address: fcollart@anl.gov.
authors.forename: Frank
authors.initials: F
authors.lastname: Collart
mesh_terms: D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D015870:Gene Expression; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012995:Solubility; D015533:Transcriptional Activation
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins
keywords: Codons; E. coli; Heterologous protein expression; Integral membrane proteins; Protein characterization; Protein sequence; Small-scale bacterial cultures; Small-scale expression of proteins