A 113-amino acid fragment of CD4 produced in Escherichia coli blocks human immunodeficiency virus-induced cell fusion.
A gene encoding a 113-amino acid, NH2-terminal fragment of CD4, rsT4.113, was constructed and expressed in Escherichia coli under the control of the tryptophan operon promoter. Following induction, rsT4.113 is produced at 5-10% of total E. coli protein, and it is found in inclusion bodies. The protein is purified in two steps under denaturing and reducing conditions. Solubilized rsT4.113 is first purified on a column of Q-Sepharose to remove low molecular weight contaminants and then purified to greater than 95% homogeneity by gel filtration. Renaturation of rsT4.113 is achieved at approximately 20% yield by dilution and dialysis. High performance liquid chromatography analysis of renatured rsT4.113 reveals a less than 15% contaminant of reduced protein. Purified and renatured rsT4.113 contains epitopes for both OKT4a and Leu3a, anti-CD4 monoclonal antibodies which block CD4-gp 120 association, but lacks measurable affinity toward a nonblocking anti-CD4 monoclonal antibody, OKT4. By comparison to a longer form (375 amino acids) of recombinant soluble T4 produced in mammalian cells that contains the entire extracellular domain, rsT4.113 has a comparable affinity for binding to OKT4a and Leu3a in a radioimmunoassay. Analysis of antiviral activity of rsT4.113 demonstrates that the E. coli-derived protein inhibits human immunodeficiency virus-induced syncytium formation with an IC50 of 5-10 micrograms/ml. These data demonstrate that the human immunodeficiency virus-binding domain of CD4 is localized within the NH2-terminal 113 amino acids of CD4 and is contained within a structure homologous to the kappa variable-like domain of immunoglobulins.
Chao
B H
BH
Biogen, Inc., Cambridge, Massachusetts 02142.
Costopoulos
D S
DS
Curiel
T
T
Bertonis
J M
JM
Chisholm
P
P
Williams
C
C
Schooley
R T
RT
Rosa
J J
JJ
Fisher
R A
RA
Maraganore
J M
JM
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000235:genetics*; D002459:Cell Fusion; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D006678:HIV / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009876:Operon; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D015728:Receptors, HIV; D011991:Receptors, Virus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D010446:Peptide Fragments; D015728:Receptors, HIV; D011991:Receptors, Virus