Screening for functional expression and overexpression of a family of diiron-containing interfacial membrane proteins using the univector recombination system.
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.
Berthold
Deborah A
DA
Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius väg 12, S-106 91 Stockholm, Sweden. berthold@dbb.su.se
Stenmark
Pål
P
Nordlund
Pär
P
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D019677:Artificial Gene Fusion; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D007501:Iron / Q000378:metabolism*; D008667:Metalloproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011995:Recombination, Genetic; D014451:Ubiquinone / Q000096:biosynthesis
D001425:Bacterial Outer Membrane Proteins; D008667:Metalloproteins; D011993:Recombinant Fusion Proteins; D014451:Ubiquinone; D007501:Iron; D010088:Oxidoreductases