Antibody fragments as inhibitors of Japanese radish acid phosphatase.
VH (heavy-chain variable region) and VL (light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and MAb-18 which inhibited Japanese radish acid phosphatase. Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar VH and identical VL domains. Initially, the VH and VL genes were expressed in Escherichia coli as single-chain Fv (ScFv) fragments. Fragments ScFv-11 and ScFv-18, named for MAb-11 and MAb-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources. ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine. The PCR-amplified VH and VL genes were subsequently expressed separately in Escherichia coli as fusion products with glutathione S-transferase. The fusion proteins had little effect on Japanese radish acid phosphatase. Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none. Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18.
Takata
R
R
Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Miyamoto
Y
Y
Honjoh
K
K
Soeda
T
T
Sakamoto
J
J
Miyamoto
T
T
Hatano
S
S
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001435:Bacteriophages; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000276:immunology*; D004926:Escherichia coli; D007128:Immunoglobulin Fragments; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D018517:Plant Roots / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000378:metabolism; D014675:Vegetables / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D000135:Acid Phosphatase