ABSTRACT
A growing number of studies require the purification of multiple proteins simultaneously and the development of simple economical high-throughput purification methods is essential. We have tested the purification of two related proteins in a variety of conditions to benchmark the semi-automated affinity chromatography method for the QIAcube that we have developed. We find that this new QIAcube method can successfully purify milligram quantities of proteins with minimal user involvement and performs as well as methods based on gravity. The method could easily be adapted to other chromatography resins and should prove to be a versatile method for optimizing protein expression or purification conditions for multiple proteins while obtaining sufficient amounts for subsequent biochemical analyses.
Subject(s)
Proteins/analysis , Proteins/chemistry , Proteomics/methods , Centrifugation/methods , Chromatography, Affinity/methods , Gene Expression , High-Throughput Screening Assays , Protein BiosynthesisABSTRACT
The Oct-1 transcription factor regulates a variety of tissue-specific and general housekeeping genes by recruiting specialized coactivators of transcription. It acts synergistically with the B-cell-specific coactivator Bob1 (OCA-B, OBF-1) to stimulate transcription of immunoglobulin genes. To analyze Oct-1's interactions with Bob1 and other regulatory proteins, we have overexpressed and purified different functional domains of the recombinant proteins. A version of Oct-1 that encompasses the amino-terminal activation region and the POU DNA-binding domain was extensively characterized (OctDeltaC1; comprising residues 1-445). Using an in vitro transcription assay, we demonstrate that this fragment is sufficient and necessary to stimulate transcription from an immunoglobulin promoter with Bob1. It also coactivates from the herpes simplex virus ICPO promoter element in the presence of VP16. Using a range of spectroscopic and biophysical techniques, we demonstrate that the activation domains of Oct-1 and Bob1 have little globular structure and that they do not physically interact. Thus, their functional synergy is likely to arise by the co-recruitment of common factors as part of a larger regulatory assembly. We propose a hypothesis to explain why the activation domains of these and other transcription factors of metazoans have little if any intrinsic structure.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Calorimetry , DNA/metabolism , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/isolation & purification , Drug Synergism , Genetic Vectors/metabolism , Host Cell Factor C1 , Humans , Molecular Sequence Data , Octamer Transcription Factor-1 , Oligodeoxyribonucleotides/metabolism , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Protein Structure, Tertiary/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Scattering, Radiation , Trans-Activators/genetics , Transcription Factors/biosynthesis , Transcription Factors/isolation & purification , Transcriptional Activation/genetics , X-RaysABSTRACT
Elongation factor-1 alpha (EF-1 alpha) is a ubiquitous, highly conserved protein that functions in peptide elongation during mRNA translation. We recently reported that, as do lower species, mammals also contain a second EF-1 alpha-like gene (S1). Unlike EF-1 alpha, which is present in all tissues, S1 mRNA is detected only in brain, heart, and muscle by Northern analysis and RNAse protection assays. In this report we present the identification of S1 and EF-1 alpha messages by non-radioactive in situ hybridization in brain and liver. We show that with this technique we can detected S1 mRNA only in certain cells in brain, mostly neurons; on the other hand, EF-1 alpha is present in all cell types that we have studied so far. We demonstrate that although EF-1 alpha mRNA can be detected in S1-negative cells it is also present in high abundance in S1-positive cells. The results presented here correlate with our previous finding that mammalian species contain a tissue-specific EF-1 alpha-like gene, S1. The presence of a second EF-1 alpha-like transcript within fully differentiated cells suggests a novel cell type-specific gene expression whose function may be related to the permanent growth-arrested state of cells in brain, heart, and muscle.
