ABSTRACT
We report two expression vectors in Pichia pastoris that direct the synthesis of recombinant single chain antibody variable region (scFv), derived from anti-Z-DNA monoclonal antibody Z22. The first vector codes for a scFv fused to the Ig binding domain of staphylococcal Protein A. The second vector codes for the scFv fused to the Fc fragment of the human IgG1. The fusion partner simplified the detection and purification of the secreted protein. These constructs yielded high level expression of an scFv with specific binding activity toward a Z form of DNA, with binding activity comparable to that of the scFv molecule produced in an Escherichia coli expression system and the original monoclonal antibody.
Subject(s)
Antigens/immunology , Immunoglobulin G/genetics , Immunoglobulin Variable Region/genetics , Pichia/genetics , Base Sequence , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Genetic Vectors , Humans , Immunoglobulin Variable Region/immunology , Oligodeoxyribonucleotides , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purificationABSTRACT
The p65 subunit of the inducible transcription factor NF-kappa B contains at least two strong transactivation domains (TADs) within its C terminus. The first domain, TA1, is contained within the last 30 amino acids of p65, whereas TA2 comprises the adjacent 90 amino acids. In this study, squelching experiments revealed that both TADs of p65, as well as the related subunit c-Rel, compete for the same cofactor(s) mediating transactivation. Both TADs of p65 share a common sequence motif, which is evolutionarily conserved and displays a remarkable degree of spatial organization when aligned on an alpha-helical surface. The functional importance of the common sequence motif was confirmed by deletion analysis of TA2. Within the conserved sequence motif, a 7-amino-acid repeat was noted. Idealized heptad repeats fused to the DNA binding domain of Gal4 were transcriptionally active, but only as multimers. Phosphorylation and transcriptional activity of a defined region within the TA2 domain was found to be stimulated by phorbol ester treatment of cells. In contrast, TA1 was constitutively phosphorylated, and its activity did not significantly respond to phorbol ester stimulation. The stimulatory effect of phorbol ester on transcription of the TA2 domain was completely blocked by the protein kinase C inhibitor. These data suggest that protein kinase C has a dual effect on NF-kappa B activity. It not only causes removal of I kappa B-alpha from cytoplasmic NF-kappa B but also augments the transactivation potential of activated nuclear NF-kappa B.
Subject(s)
NF-kappa B/chemistry , Tetradecanoylphorbol Acetate/pharmacology , Transcriptional Activation , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Molecular Sequence Data , NF-kappa B/metabolism , Phosphorylation , Protein Structure, Secondary , Transcription Factor RelAABSTRACT
The p65 subunit of the NF-kappa B transcription factor contains in its C-terminal 120 amino acids at least two transcription activation domains. One domain (TA1) is contained within only the 30 C-terminal amino acids. Structural studies employing CD and NMR spectroscopy revealed that the TA1 domain is unstructured. NMR analysis of a protein corresponding to the C-terminal 123 amino acids also showed a random coil conformation. However, a portion of TA1 was found to adopt an alpha-helical conformation in the presence of hydrophobic solvents. Transcriptional analysis of point mutants revealed the functional importance of two evolutionary conserved sequence repeats, which are located in the conditionally alpha-helical region of TA1. These repeats acted synergistically in transcription activation. The inhibitory effect of some mutants indicated secondary structure constraints on TA1 in intact cells. Inverting the sequence of two acidic activation domains significantly reduced their transactivating potential, suggesting that amino acid composition is not solely essential for activity; a defined primary structure is necessary as well. Acidic sequence motifs related in primary structure and squelching activity to those of TA1 are present in the activation domains of VP16, c-Rel, and several other transcription factors. We propose a model suggesting that primarily unstructured acidic activation domains can adopt a secondary structure upon contacting their target molecules by an "induced fit" mechanism.
