Expression, purification, and membrane reconstitution of a CD4 fragment comprising the transmembrane and cytoplasmic domains of the receptor.
The transmembrane glycoprotein CD4 plays a prominent role in the adaptive immune response. CD4 is displayed primarily on the surface of T helper cells, but also on subsets of memory and regulatory T lymphocytes, macrophages, and dendritic cells. Binding of the lymphocyte specific tyrosine kinase p56(lck) to the cytoplasmic domain of CD4 is crucial for antigen receptor-mediated signal transduction. The human immunodeficiency virus (HIV) utilizes CD4 as the main receptor for T cell invasion. The virus has developed multiple strategies for down-regulation of CD4 in infected cells. Physical interactions of viral proteins VpU and Nef with the cytoplasmic tail of CD4 initiate a cascade of events leading to degradation of CD4. Here we report heterologous expression and purification of a CD4 fragment comprising the transmembrane and cytoplasmic domains of human CD4. A synthetic gene encoding CD4 amino acid residues 372-433 and a protease cleavage site was cloned into the pTKK19xb/ub plasmid. The CD4 fragment was expressed in Escherichia coli C43(DE3) cells as a ubiquitin fusion with an N-terminal His-tag, isolated, released by PreScission proteolytic cleavage, and purified to homogeneity. Incorporation of the recombinant CD4 fragment in lipid membranes and physical interaction with the cytoplasmic domain of VpU was demonstrated by centrifugation assays followed by reversed phase chromatographic analysis of the composition of the proteoliposomes. A high resolution NMR spectrum of uniformly (15)N-labeled CD4 peptide in membrane simulating micelles proves the possibility of solution NMR studies of this CD4 fragment and of its molecular complexes.
Wittlich
Marc
M
Institut für Physikalische Biologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.
Wiesehan
Katja
K
Koenig
Bernd W
BW
Willbold
Dieter
D
D000595:Amino Acid Sequence; D015704:CD4 Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002462:Cell Membrane / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry
D015704:CD4 Antigens; D008081:Liposomes; D011994:Recombinant Proteins; D054334:Viral Regulatory and Accessory Proteins