title: A new expression vector facilitating production and functional analysis of scFv antibody fragments selected from Tomlinson I+J phagemid libraries.
abstract: Tomlinson I+J are synthetic phagemid human scFv libraries widely employed to obtain specific antibody fragments via a phage display method. The pIT2/HB2151 expression system proposed by the designers of the libraries has certain drawbacks which result in the lack of expression or low expression levels of numerous soluble scFvs. At the stage of scFv screening, this may lead to losing some excellent antibodies, which can be avoided but requires laborious and expensive work. Here we present a new, pET-30-based vector, which is compatible with Tomlinson libraries, retains all virtues of pIT2 used as a plasmid and eliminates all its flaws. We demonstrate that pET-scFv-T is frequently superior to pIT2 in terms of efficient scFv expression. Moreover, an amber suppressor bacterial strain, RosettaBlue(DE3)pLysS, transformed with the new vector, pET-scFv-T, coding for a number of scFvs, produces substantial amounts of functional, easy to purify recombinant antibody fragments, regardless of whether their coding sequences contain amber codons. Thus, pET-scFv-T/RosettaBlue(DE3)pLysS expression system seems to be a perfect tool for screening for the finest soluble scFvs selected from Tomlinson I+J, as well as from many other phagemid libraries.
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: karolina.ossysek@uj.edu.pl.
authors.forename: Karolina
authors.initials: K
authors.lastname: Ossysek
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: tomasz.uchanski@vib-vub.be.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Uchański
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: malgorzata.kulesza@doctoral.uj.edu.pl.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Kulesza
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland; Malopolska Centre of Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7A, 30-387 Kraków, Poland. Electronic address: monika.bzowska@uj.edu.pl.
authors.forename: Monika
authors.initials: M
authors.lastname: Bzowska
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland; Malopolska Centre of Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7A, 30-387 Kraków, Poland. Electronic address: tomasz.klaus@doctoral.uj.edu.pl.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Klaus
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: klaudia.wos@uj.edu.pl.
authors.forename: Klaudia
authors.initials: K
authors.lastname: Woś
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: mariusz.madej@uj.edu.pl.
authors.forename: Mariusz
authors.initials: M
authors.lastname: Madej
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: joanna.bereta@uj.edu.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Bereta
mesh_terms: D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016366:Open Reading Frames; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D003062:Codon; D019151:Peptide Library; D057127:Single-Chain Antibodies
keywords: Amber codon; Expression vector; Tomlinson I+J; scFv