Stability studies of nucleic acid-binding Fab isolated from combinatorial bacteriophage display libraries.
This study examined the global stability and activity properties of recombinant DNA-binding antibody fragments that were obtained from a bacteriophage combinatorial display library. The goal of this study was to determine whether the combinatorial approach of heavy and light chain assembly in E. coli and subsequent affinity selection preferentially selects for antibody fragments with unusual structural stabilities. Specifically, the binding properties and stability of recombinant antibody fragments with or without a C-terminal His tag to temperature, pH, and guanidine-HCI were examined. Both Fab exhibited almost identical Kd (120-130, 140-170, and 450-560 nM) and maximal fluorescence quenching (20-25%) values for binding to (dT)20, (dT)15, and (dT)10, respectively. Thermal denaturation data obtained by CD spectroscopy demonstrated that both Fab possessed structural properties comparable to well-folded proteins with defined tertiary structures which were stable below 70 degrees C (Tm 73 degrees C). These results were confirmed by differential scanning calorimetry. Both Fab exhibited the same rate of irreversible thermal inactivation (0.061-0.069 min-1) at 75 degrees C and could be reversibly renatured from guanidine-HCI and pH extremes. Crystallization trials with one recombinant DNA-binding Fab yielded diffraction quality crystals also suggesting a well-defined tertiary structure.
Deutscher
S L
SL
Department of Biochemistry, University of Missouri at Columbia, Missouri 65211, USA.
Crider
M E
ME
Ringbauer
J A
JA
Komissarov
A A
AA
Quinn
T P
TP
D000818:Animals; D003090:Coliphages / Q000235:genetics; D003460:Crystallization; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D015394:Molecular Structure; D009696:Nucleic Acids / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D007140:Immunoglobulin Fab Fragments; D009696:Nucleic Acids; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C027903:oligo (dT)