RESUMO
The complementarity-determining regions (CDRs) of a human kappa light chain were replaced with CDRs from a murine gamma-1 heavy chain and, by use of molecular modeling, key heavy chain framework residues were identified and thus included to preserve the native conformation of the heavy chain CDRs. Co-expression of this hybrid human kappa chain (V[HB]C[L]) with a human kappa chain counterpart (V[L]C[L], engineered to contain murine light chain CDRs) resulted in the secretion of high levels of a heterodimeric protein (V[HB]C[L]::V[L]C[L]) termed 'kappabody'. This protein also had equivalent affinity for antigen as the Fab' of the parent murine IgG1. High-level secretion was also observed for the hybrid chain as homodimers (V[HB]C[L]::V[HB]C[L]), which is not observed for chimeric chains consisting of a heavy chain variable region and light chain constant region, i.e. V[H]C[L] homodimers or single chains are not secreted. This indicates that regions within the variable domain, required for secretion of light chains, reside outside of the hypervariable regions (CDRs) and that the heavy chain CDRs and supporting residues do not prevent secretion. These results demonstrate the possibility of designing small, single-domain molecules possessing a given binding activity which may be secreted at high levels from mammalian cells.
Assuntos
Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Ligação Competitiva , Dimerização , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Humanos , Hibridomas , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/genética , Cinética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Análise de Sequência , TransfecçãoRESUMO
We define a protein complex present in avian nuclear extracts that interacts with the Schmidt-Ruppin strain of the Rous sarcoma virus (RSV) long terminal repeat (LTR) between positions -197 and -168 relative to the transcriptional start site. We call this complex EFIV and demonstrate that the EFIV protein(s) is present in several avian cell types examined, including B cells (S13 and DT40), T cells (MSB), and chicken embryo fibroblasts. We also report that the EFIV binding site activates transcription of reporter constructs after transfection into avian B cells and chicken embryo fibroblasts, demonstrating that the EFIV region constitutes a functional transactivator sequence. By chemical interference footprinting and mutational analyses we define the EFIV binding site as including the sequence GCAACATG, which is present in two copies between positions -197 and -168, as well as sequences that lie between the two repeats. Electrophoretic mobility shift competition experiments suggest that the EFIV protein(s) may be related to members of the CCAAT/enhancer-binding protein family of transcription factors that interact with different regions of the RSV and the avian leukosis virus (ALV) LTRs. However, as defined by differences in sensitivity to protein synthesis inhibitors and footprinting patterns, EFIV is clearly distinct from these previously defined LTR binding factors. In addition, the finding that EFIV binding activity is stable in B cells indicates either that the lability of all 5' LTR binding activities is not required for B-cell transformation by the ALV/RSV family of viruses or that nonacute transforming viruses that include an RSV LTR may use a mechanism to effect cellular transformation different from that proposed for ALV.
