Design and expression of soluble CTLA-4 variable domain as a scaffold for the display of functional polypeptides.
We have designed and engineered the human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) variable (V-like) domain to produce a human-based protein scaffold for peptide display. First, to test whether the CTLA-4 CDR-like loops were permissive to loop replacement/insertion we substituted either the CDR1 or CDR3 loop with somatostatin, a 14-residue intra-disulfide-linked neuropeptide. Upon expression as periplasmic-targeted proteins in Escherichia coli, molecules with superior solubility characteristics to the wild-type V-domain were produced. These mutations in CTLA-4 ablated binding to its natural ligands CD80 and CD86, whereas binding to a conformation-dependent anti-CTLA-4 monoclonal antibody showed that the V-domain framework remained correctly folded. Secondly, to develop a system for library selection, we displayed both wild-type and mutated CTLA-4 proteins on the surface of fd-bacteriophage as fusions with the geneIII protein. CTLA-4 displayed on phage bound specifically to immobilized CD80-Ig and CD86-Ig and in one-step panning enriched 5,000 to 2,600-fold respectively over wild-type phage. Bacteriophage displaying CTLA-4 with somatostatin in CDR3 (CTLA-4R-Som3) specifically bound somatostatin receptors on transfected CHO-K1 cells pre-incubated with 1 microg/ml tunicamycin to remove receptor glycosylation. Binding was specific, as 1 microM somatostatin successfully competed with CTLA-4R-Som3. CTLA-4R-Som3 also activated as well as binding preferentially to non-glycosylated receptor subtype Sst4. The ability to substitute CDR-like loops within CTLA-4 will enable design and construction of more complex libraries of single V-like domain binding molecules. Proteins 1999;36:217-227.
Nuttall
S D
SD
CRC for Diagnostic Technologies and CSIRO Molecular Science, Parkville, Victoria, Australia. Stewart.Nuttall@molsci.csiro.au
Rousch
M J
MJ
Irving
R A
RA
Hufton
S E
SE
Hoogenboom
H R
HR
Hudson
P J
PJ
D000069594:Abatacept; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D000943:Antigens, Differentiation / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018122:B7-1 Antigen / Q000378:metabolism; D051940:B7-2 Antigen; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D016466:CHO Cells; D060908:CTLA-4 Antigen; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D018796:Immunoconjugates; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D017481:Receptors, Somatostatin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013004:Somatostatin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018122:B7-1 Antigen; D051940:B7-2 Antigen; C497964:CD86 protein, human; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D018796:Immunoconjugates; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019151:Peptide Library; D010455:Peptides; D017481:Receptors, Somatostatin; D011993:Recombinant Fusion Proteins; C098681:somatostatin receptor subtype-4; D013004:Somatostatin; D000069594:Abatacept