Cloning and expression of the variable regions of mouse myeloma protein MOPC315 in E. coli: recovery of active FV fragments.
Recombinant DNA techniques were used to clone and express the FV portion of MOPC315, a mouse myeloma protein with a high affinity for 2,4-dinitrophenyl (DNP). The FV fragment consists of a heterodimer of heavy and light chain variable domains (VH and VL). Two separate bacterial plasmid constructs, containing either a variable region cDNA for the light chain or a variable region synthetic gene for the heavy chain demonstrated high levels of expression (150-200 mg/L) under control of the bacteriophage T7 promoter. Recombinant chains were initially recovered as inclusion bodies and then dissolved separately in 8 M urea, combined together, and refolded by subsequent chaotrope removal. Biologically active FV was affinity purified from the chain mixture by specific binding to DNP-lysine-Sepharose. Yields of active material as high as 20% were obtained with activity confirmed by fluorescence quench analysis. The purified FV displayed a binding affinity of 4.8 +/- 0.3 x 10(-7) M which was identical to the native FV. Chimeric FVs composed of recombinant and native chain mixtures yielded similar results. Recombinant MOPC315 FV activity was also obtained using a single chain construct (sFV), in which recombinant VH and VL were linked via a (Gly4Ser)3 spacer region. Binding affinity of the sFV was shown to be the same as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 FV makes this system useful in the study of the optimization of antibody production in bacteria.
Cheadle
C
C
Rhône-Poulenc Rorer Central Research, King of Prussia, PA 19406.
Hook
L E
LE
Givol
D
D
Ricca
G A
GA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004136:Dinitrobenzenes / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli; D005803:Genes, Immunoglobulin; D007070:Immunoglobulin A / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009194:Myeloma Proteins / Q000235:genetics*; D009841:Oligonucleotides / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
D004136:Dinitrobenzenes; D007070:Immunoglobulin A; D007135:Immunoglobulin Variable Region; D009194:Myeloma Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins