Regulated secretion and purification of recombinant antibodies in E. coli.
A plasmid for optimized protein expression of recombinant Fv antibodies (pOPE) in E. coli was used to express the variable domains of the murine monoclonal antibody HD39 specific for the human B-cell surface antigen CD22. The production of Fv antibodies by pOPE can be regulated over a wide range by varying the IPTG concentration. Antibodies that can discriminate between secreted and nonsecreted Fv antibody fragments were used to show that secretion is the limiting step for the production of functional Fv antibodies. IPTG concentrations above 20 microM increased the total antibody production, but did not yield larger amounts of secreted Fv antibodies. The addition of five histidines to the C terminus facilitates an easy single-step enrichment procedure based on immobilized metal affinity chromatography.
Dübel
S
S
Recombinant Antibody Group, German Cancer Research Center, Heidelberg.
Breitling
F
F
Klewinghaus
I
I
Little
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D000918:Antibody Specificity; D015703:Antigens, CD / Q000032:analysis / Q000276:immunology; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D015815:Cell Adhesion Molecules; D002846:Chromatography, Affinity; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000276:immunology / Q000378:metabolism*; D005822:Genetic Vectors; D006639:Histidine / Q000494:pharmacology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology; D037102:Lectins; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D051918:Sialic Acid Binding Ig-like Lectin 2
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D000954:Antigens, Surface; D001426:Bacterial Proteins; C497867:CD22 protein, human; C497868:Cd22 protein, mouse; D015815:Cell Adhesion Molecules; D004269:DNA, Bacterial; D007128:Immunoglobulin Fragments; D037102:Lectins; D011994:Recombinant Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2; C055966:immunoglobulin Fv; D006639:Histidine