Preparation and crystallization of a human immunodeficiency virus p24-Fab complex.
A recombinant form of human immunodeficiency virus capsid protein, p24, expressed in Escherichia coli has been purified to homogeneity and separated into distinct isoelectric forms. A monoclonal antibody, mAb25.4, which recognizes an epitope in the amino-terminal region of p24, has been purified to homogeneity from ascites fluid and digested with papain to produce the respective antigen-binding fragment (Fab). The Fab25.4 was purified from the digestion mixture and separated into two distinct isoelectric forms. The two Fab species were each complexed with one isoelectric form of the recombinant p24 by incubating equimolar quantities of the two proteins. Two different crystal morphologies of the p24-Fab25.4 complex were obtained by the vapor-diffusion method with 12-24% PEG 3350 as the precipitant. One of these crystal forms has unit-cell parameters of a = 92.1 A, b = 85.4 A, c = 54.0 A, alpha = gamma = 90.0 degrees and beta = 90.4 degrees and belongs to the monoclinic space group P2(1), with one molecule of the complex per asymmetric unit. These crystals strongly diffracted x-rays to at least 2.7-A resolution.
Prongay
A J
AJ
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.
Smith
T J
TJ
Rossmann
M G
MG
Ehrlich
L S
LS
Carter
C A
CA
McClure
J
J
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002213:Capsid / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015683:Gene Products, gag / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006678:HIV / Q000235:genetics*; D016655:HIV Core Protein p24; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D014758:Viral Core Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D014961:X-Ray Diffraction
D000911:Antibodies, Monoclonal; D015683:Gene Products, gag; D016655:HIV Core Protein p24; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D014758:Viral Core Proteins