HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Berthold, D. A. Articles by Nordlund, P. Search for Related Content PubMed PubMed Citation Articles by Berthold, D. A. Articles by Nordlund, P. Social Bookmarking                   What's this?

Screening for functional expression and overexpression of a family of diiron-containing interfacial membrane proteins using the univector recombination system

Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius väg 12, S-106 91 Stockholm, Sweden

Reprint requests to: Deborah Berthold, Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius väg 12, S-106 91 Stockholm, Sweden; e-mail: berthold{at}dbb.su.se; fax: 46 8 15 3679.

The large number of uncharacterized genes emerging from genome sequencing projects has resulted in a need for quick and reliable screening methods for protein expression parameters. We have utilized the univector plasmid recombination system (as previously reported) to develop a series of vectors for rapid screening for expression in Escherichia coli. A high level of recombinant protein expression is a requirement for purification of protein for structural determination and other purposes. In other applications, successful complementation of a missing enzyme activity in E. coli, as well as directed evolution studies and metabolic engineering, often require a much lower level of protein expression. In this report we describe the construction of a number of new pHOST vectors that can be screened for both low- and high-level expression. We isolated a mutant vector for MBP fusions that exhibited a more optimal level of expression for complementation of aerobic respiration in hemA^- E. coli, our functional assay for the alternative oxidase. We then demonstrated the use of our system to rapidly screen for both optimal functional expression and optimal overexpression of the alternative oxidase as well as two other members of a family of membrane-bound diiron carboxylate proteins, the plastid terminal oxidase and 5-demethoxyquinone hydroxylase.

Keywords: Di-iron proteins; membrane proteins; protein expression; alternative oxidase; clk-1; Coq7; IMMUTANS; ubiquinone biosynthesis

Abbreviations: AOX, alternative oxidase • Coq7, 5-demethoxyquinone hydroxylase • IPTG, isopropylthio-ß-D-galactoside • MBP, maltose binding protein • pHOST, a generic term in the univector system for an expression vector containing an in-frame loxP site • pUNI, the generic term in the univector system for the entry vector (into which a coding sequence is inserted) • PTOX, plastid terminal oxidase

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				G. Marsischky and J. LaBaer Many Paths to Many Clones: A Comparative Look at High-Throughput Cloning Methods Genome Res., October 1, 2004; 14(10b): 2020 - 2028. [Abstract] [Full Text] [PDF]