Overexpression inEscherichia coli,Purification, and Characterization of Recombinant 60S Ribosomal Acidic Proteins fromSaccharomyces cerevisiae

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• 作者：Tchó ; rzewski ; Marek ; Boguszewska ; Aleksandra ; Abramczyk ; Dariusz ; Grankowski ; Nikodem
• 刊名：Protein Expression and Purification
• 出版年：1999
• 出版时间：February, 1999
• 年：1999
• 卷：15
• 期：1
• 页码：40-47
• 全文大小：160 K

文摘

The 60S ribosomal subunits fromSaccharomyces cerevisiaecontain a set of four acidic proteins named YP1α, YP1β, YP2α, and YP2β. The genes for each were PCR amplified from a yeast cDNA library, sequenced, and expressed inEscherichia colicells using two expression systems. The first system, pLM1, was used for YP1β, YP2α, and YP2β. The second one, pT7-7, was used for YP1α. 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 (YP1α, YP1β) or NH₄Cl/ethanol extraction (YP2α, YP2β). 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 YP2α and YP2β showed the pIs of the recombinant proteins are the same as that of the native yeast ribosomal P2 proteins. The pIof YP1α is changed due to the addition of five amino acids attached to the N-terminus of recombinant polypeptide from the expression vector. YP1β was obtained as a truncated form of polypeptide, similar to its ribosomal counterpart, YP1β′. This was proved by isoelectrofocusing gel analysis.