Parthun M R, Mangus D A, Jaehning J A
Department of Biology, Indiana University, Bloomington 47405.
Mol Cell Biol. 1992 Dec;12(12):5683-9. doi: 10.1128/mcb.12.12.5683-5689.1992.
A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose.
包括滤膜结合、足迹法和凝胶阻滞在内的多种技术，可用于在从酵母细胞中初步纯化转录激活因子GAL4（Gal4p）的过程中对其进行检测。经过DNA亲和层析后，通过滤膜结合或足迹法检测发现Gal4p仍能选择性地结合DNA。然而，通过凝胶阻滞检测发现，亲和纯化后的蛋白不再能够与DNA形成稳定的复合物。将纯化的Gal4p与DNA亲和柱的流出组分混合，可恢复凝胶阻滞复合物的形成。凝胶阻滞检测用于监测从亲和柱流出物中纯化热稳定的Gal4p-DNA复合物稳定活性的过程。该活性在最终纯化步骤中与21 kDa和27 kDa的多肽共洗脱。根据其N端氨基酸序列克隆了编码21 kDa蛋白的酵母基因。该基因名为EGD1（GAL4 DNA结合增强子），编码一种高度碱性的蛋白（21%为赖氨酸和精氨酸），预测分子量为16.5 kDa。EGD1产物Egd1p的氨基酸序列与人类蛋白BTF3的序列高度相似（X.M.郑、D.布莱克、P.尚邦和J.M.埃格利，《自然》[伦敦]344:556 - 559，1990）。尽管egd1缺失突变体是有活力的且为Gal+，但当细胞从葡萄糖转移到半乳糖时，egd1突变体菌株中半乳糖调节基因的诱导显著降低。
