Reconstitution and purification of flagellar filaments from Caulobacter crescentus

Filaments from isolated flagella of Caulobacter crescentus have been purified by successive dissociation and reconstitution. After the second and third reconstitutions from subunits in 0.8 M sodium citrate, filament preparations contained only two proteins, flagellin A (26,000 daltons) and flagellin B (28,000 daltons). There was some enrichment for flagellin A during reconstitution by this procedure, since isolated flagella contained flagellin A and flagellin B in a ratio of approximately 3.8:1 and filaments after the third reconstitution contained the two proteins in a ratio of 5.0:1.</jats:p

Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.</jats:p

Purification of multiple erythroid cell proteins that bind the promoter of the alpha-globin gene.

Three erythroid cell factors that bind the murine alpha-globin promoter were enriched more than 1,000-fold by conventional and DNA sequence affinity chromatography. Visualization of enriched polypeptides revealed simple patterns suggesting that each binding activity was purified. Two of the purified proteins, alpha-CP1 and alpha-CP2, have been shown previously to interact with distinct binding sites that overlap in the alpha-globin CCAAT box. Affinity purification of alpha-CP1 revealed seven polypeptides with Mrs raging from 27,000 to 38,000. In contrast, purified alpha-CP2 was made up of a polypeptide doublet with Mrs of 64,000 and 66,000. The third purified binding activity, alpha-IRP, interacted with sequences that formed an inverted repeat (IR) between the alpha-globin CCAAT and TATAA boxes. Affinity-purified alpha-IRP was made up of a single polypeptide with an Mr of 85,000. We confirmed that the purified polypeptides corresponded to alpha-CP1-, alpha-CP2-, and alpha-IRP-binding activities by UV cross-linking experiments (alpha-CP2 and alpha-IRP) or by renaturation of binding activity after elution of polypeptides from sodium dodecyl sulfate-polyacrylamide gels (alpha-CP1 and alpha-CP2). The apparent complexity of the polypeptides accounting for alpha-CP1 binding activity prompted a further physical characterization of this factor. Sedimentation of affinity-purified alpha-CP1 in glycerol gradients containing 100 mM KCl showed that all seven polypeptides migrated as a complex that cosedimented with alpha-CP1-binding activity. In contrast, when sedimented in glycerol gradients containing 500 mM KCl, alpha-CP1 dissociated into at least two components. Under these conditions, alpha-CP1-binding activity was reduced or lost. Activity was reconstituted, however, by combining fractions that were enriched in the two components. These results were confirmed by experiments in which we showed that alpha-CP1-binding activity can be recovered only by combining distinct sets of polypeptides that were isolated and renatured from sodium dodecyl sulfate-polyacrylamide gels. Our results suggest that the seven polypeptides visualized after affinity purification of alpha-CP1 interact to form a heterotypic complex (or set of complexes) required for alpha-CP1-binding activity

Molecular cloning of the alpha-globin transcription factor CP2

CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1.</jats:p