High level expression of a protein precursor for functional studies.
In vitro analyses of type I signal peptidase activities require protein precursors as substrates. Usually, these pre-proteins are expressed in vitro and cleavage of the signal sequence is followed by SDS polyacrylamide gel electrophoresis coupled with autoradiography. Radioactive amino acids have to be incorporated in the expressed protein, since the amount of the in vitro expressed protein is usually very low and processing of the signal peptide cannot be followed by SDS polyacrylamide gel electrophoresis alone. Here we describe a rapid and simple method to express large amounts of a protein precursor in E. coli. We have analyzed the effect of ionophors as well as of azide on the accumulation of expressed protein precursors. Azide blocks the function of SecA and the ionophors dissipate the electrochemical gradient across the cytoplasmic membrane of E. coli. Addition of azide ions resulted in the formation of inclusion bodies, highly enriched with pre-apo-plastocyanine. Plastocyanine is a soluble copper protein, which can be found in the periplasmic space of cyanobacteria as well as in the thylakoid lumen of cyanobacteria and chloroplasts, and the pre-protein contains a cleavable signal sequence at its N-terminus. After purification of cyanobacterial preapo-plastocyanine, its signal sequence can be cleaved off by the E. coli signal peptidase, and protein processing was followed on Coomassie stained SDS polyacrylamide gels. We are optimistic that the presented method can be further developed and applied.
Gathmann
Sven
S
Institut fur Biochemie und Molekularbiologie, Albert-Ludwigs-Universitat Freiburg, Stefan-Meier-Strasse 19, 79104 Freiburg, Germany.
Rupprecht
Eva
E
Schneider
Dirk
D
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015870:Gene Expression; D010957:Plasmids / Q000235:genetics; D010970:Plastocyanin / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000502:physiology*
D001059:Apoproteins; D001386:Azides; D011498:Protein Precursors; D021382:Protein Sorting Signals; C040778:apoplastocyanin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D010970:Plastocyanin