Bustin M, Becerra P S, Crippa M P, Lehn D A, Pash J M, Shiloach J
Laboratory of Molecular Carcinogenesis, NCI National Institutes of Health, Bethesda, MD 20892.
Nucleic Acids Res. 1991 Jun 11;19(11):3115-21. doi: 10.1093/nar/19.11.3115.
Vectors for expressing human chromosomal proteins HMG-14 and HMG-17 in bacterial cultures under the control of the temperature-inducible lambda PL promoter have been constructed. The open reading frames of the cDNAs have been amplified by the polymerase chain reaction (PCR), utilizing amplimers containing desired restriction sites, thereby facilitating precise location of the initiation codon downstream from a ribosomal binding site. Expression of the recombinant proteins does not significantly affect bacterial growth. The rate of synthesis of the recombinant proteins is maximal during the initial stages of induction and slows down appreciably with time. After an initial burst of protein synthesis, the level of the recombinant protein in the bacterial extracts remains constant at different times following induction. Methods for rapid extraction and purification of the recombinant proteins are described. The recombinant proteins are compared to the proteins isolated from eucaryotic cells by electrophoretic mobility, Western analysis and nucleosome core mobility-shift assays. The ability of the proteins to shift the mobility of the nucleosome cores, but not that of DNA, can be used as a functional assay for these HMG proteins. A source for large quantities of human chromosomal proteins HMG-14 and HMG-17 will facilitate studies on their structure, cellular function and mechanism of interaction with nucleosomes.
已构建出在温度诱导型λPL启动子控制下在细菌培养物中表达人染色体蛋白HMG - 14和HMG - 17的载体。利用含有所需限制性位点的扩增引物，通过聚合酶链反应（PCR）扩增cDNA的开放阅读框，从而便于起始密码子精确位于核糖体结合位点下游。重组蛋白的表达不会显著影响细菌生长。重组蛋白的合成速率在诱导初期最大，随时间明显减慢。在蛋白质合成的初始爆发后，诱导后不同时间细菌提取物中重组蛋白的水平保持恒定。描述了重组蛋白的快速提取和纯化方法。通过电泳迁移率、蛋白质免疫印迹分析和核小体核心迁移率变动分析，将重组蛋白与从真核细胞中分离的蛋白进行比较。这些蛋白使核小体核心迁移率发生改变而不影响DNA迁移率的能力，可作为这些HMG蛋白的功能测定方法。大量人染色体蛋白HMG - 14和HMG - 17的来源将有助于对其结构、细胞功能以及与核小体相互作用机制的研究。
