title: Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant.
abstract: Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.
authors.affiliation: a Department of Clinical Medicine and Sino-Danish Center ; Aarhus University ; Aarhus , Denmark ;
authors.forename: Simon
authors.initials: S
authors.lastname: Lykkemark
authors.forename: Ole Aalund
authors.initials: OA
authors.lastname: Mandrup
authors.forename: Niels Anton
authors.initials: NA
authors.lastname: Friis
authors.forename: Peter
authors.initials: P
authors.lastname: Kristensen
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000906:Antibodies; D007074:Immunoglobulin G; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
keywords: DNA, deoxyribonucleic acid; E. coli, escherichia coli; HRP, horseradish peroxidase; IPTG, isopropyl β-D-1-thiogalactopyranoside; PCR, polymerase chain reaction; RCF, relative centrifugal force; TEV protease, tobacco etch virus; Tsp protease, tail-specific protease; antibodies; dAb, domain antibody; peptide tags; phage display; protein expression; proteolytic degradation; rpm, revolutions per minute; scFv, single chain fragment variable