Tchórzewski M, Boguszewska A, Abramczyk D, Grankowski N
Department of Molecular Biology, Maria Curie-Sklodowska University, Institute of Microbiology and Biotechnology, Akademicka 19 Street, Lublin, 20-033, Poland.
Protein Expr Purif. 1999 Feb;15(1):40-7. doi: 10.1006/prep.1998.0997.
The 60S ribosomal subunits from Saccharomyces cerevisiae contain a set of four acidic proteins named YP1alpha, YP1beta, YP2alpha, and YP2beta. The genes for each were PCR amplified from a yeast cDNA library, sequenced, and expressed in Escherichia coli cells using two expression systems. The first system, pLM1, was used for YP1beta, YP2alpha, and YP2beta. The second one, pT7-7, was used for YP1alpha. Expression in both cases was under the control of a strong inducible T7 promoter. The amount of induced recombinant proteins in the host cells was around 10 to 20% of the total soluble bacterial proteins. A new protocol for purification of all four recombinant proteins was established. The preliminary steps of purification were done by ammonium sulfate precipitation (YP1alpha, YP1beta) or NH4Cl/ethanol extraction (YP2alpha, YP2beta). The recombinant proteins were then purified to apparent homogeneity by only two steps of classical chromatographies, ion exchange (DEAE-cellulose) and gel filtration (Sephacryl S-200). Isoelectrofocusing analysis of YP2alpha and YP2beta showed the pIs of the recombinant proteins are the same as that of the native yeast ribosomal P2 proteins. The pI of YP1alpha is changed due to the addition of five amino acids attached to the N-terminus of recombinant polypeptide from the expression vector. YP1beta was obtained as a truncated form of polypeptide, similar to its ribosomal counterpart, YP1beta'. This was proved by isoelectrofocusing gel analysis.
酿酒酵母的60S核糖体亚基包含一组四种酸性蛋白，分别命名为YP1α、YP1β、YP2α和YP2β。通过PCR从酵母cDNA文库中扩增出每种蛋白的基因，进行测序，并使用两种表达系统在大肠杆菌细胞中进行表达。第一个系统pLM1用于YP1β、YP2α和YP2β。第二个系统pT7 - 7用于YP1α。两种情况下的表达均受强诱导型T7启动子的控制。宿主细胞中诱导的重组蛋白量约占细菌总可溶性蛋白的10%至20%。建立了一种纯化所有四种重组蛋白的新方案。纯化的初步步骤通过硫酸铵沉淀（YP1α、YP1β）或NH4Cl/乙醇提取（YP2α、YP2β）进行。然后仅通过两步经典色谱法，即离子交换（DEAE - 纤维素）和凝胶过滤（Sephacryl S - 200），将重组蛋白纯化至表观均一。对YP2α和YP2β的等电聚焦分析表明，重组蛋白的等电点与天然酵母核糖体P2蛋白相同。由于表达载体中重组多肽N端添加了五个氨基酸，YP1α的等电点发生了变化。YP1β是以截短形式的多肽获得的，类似于其核糖体对应物YP1β'。这通过等电聚焦凝胶分析得到了证实。
