title: A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F(ab) fragments and leads to improved recombinant immunoglobulin stability.
abstract: Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.
authors.affiliation: Department fur Pflanzenwissenschaften, Lehrstuhl fur Botanik, Wissenschaftszentrum Weihenstephan fur Ernahrung, Landnutzung und Umwelt, Technische Universitat Munchen, Alte Akademie 12, D-85350, Freising-Weihenstephan, Germany. kramer@wzw.tum.de
authors.forename: Karl
authors.initials: K
authors.lastname: Kramer
authors.forename: Markus
authors.initials: M
authors.lastname: Fiedler
authors.forename: Arne
authors.initials: A
authors.lastname: Skerra
authors.forename: Bertold
authors.initials: B
authors.lastname: Hock
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D007128:Immunoglobulin Fragments / Q000235:genetics; D051379:Mice; D008957:Models, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv