Overexpression in Escherichia coli, purification, and characterization of recombinant 60S ribosomal acidic proteins from Saccharomyces cerevisiae

The 60S ribosomal subunits from Saccharomyces cerevisiae contain a set of four acidic proteins named YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta. 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 YP1 beta, YP2 alpha, and YP2 beta. The second one, pT7-7, was used for YP1 alpha. 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 (YP1 alpha, YP1 beta) or NH4Cl/ethanol extraction (YP2 alpha, YP2 beta). 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). Isoelectrofocussing analysis of YP2 alpha and YP2 beta showed the pls of the recombinant proteins are the same as that of the native yeast ribosomal P2 proteins. The pi of YP1 alpha is changed due to the addition of five amino acids attached to the N-terminus of recombinant polypeptide from the expression vector. YP1 beta was obtained as a truncated form of polypeptide, similar to its ribosomal counterpart, YP1 beta'. This was proved by isoelectrofocusing gel analysis. (C) 1999 Academic Press.