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.
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.
Kramer
Karl
K
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
Fiedler
Markus
M
Skerra
Arne
A
Hock
Bertold
B
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
D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv