Staphylococcal protein A as a fusion partner directs secretion of the e1alpha and e1beta subunits of pea mitochondrial pyruvate dehydrogenase by Bacillus subtilis.
Staphylococcal protein A (SPA)-based vectors were constructed to direct secretion of the E1alpha and E1beta subunits of Pisum sativum mitochondrial pyruvate dehydrogenase from Bacillus subtilis. These proteins were not exported when the signal peptide from levansucrase (SacBSP) was fused to their N-termini. Both SacBSP-E1alpha and SacBSP-E1beta fusion proteins were insoluble in the cytoplasm. However, when the SPA open-reading frame was inserted between SacBSP and E1alpha or E1beta, corresponding fusion proteins were secreted from the cells. The first (E) IgG-binding domain of SPA was sufficient to direct low level secretion of both fusion proteins (SacBSP-E-E1alpha and SacBSP-E-E1beta). Adding the second (D) IgG-binding domain improved extracellular protein yields 3- to 4-fold over E alone, but was not as efficient as secretion of the full-length (EDABC) SPA-fusion proteins. All constructs were based on the pUB110-derived multicopy plasmid pWB705. Separate B. subtilis strains transformed with SacBSP-E-E1alpha-His(6) or SacBSP-E1beta were cocultivated in the presence of Ni-NTA agarose. The native pyruvate dehydrogenase alpha2beta2 structure was bound to the affinity matrix, demonstrating assembly after secretion. The use of SPA as a fusion partner during expression of heterologous proteins by B. subtilis provides the basis of a versatile system that can be used to study both secretion and protein:protein interactions.
Moreno
J I
JI
Biochemistry Department, University of Missouri, Columbia, Missouri 65211, USA.
Miernyk
J A
JA
Randall
D D
DD
D000818:Animals; D001412:Bacillus subtilis / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics; D005822:Genetic Vectors; D008928:Mitochondria / Q000201:enzymology*; D018532:Pisum sativum / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D030481:Pyruvate Dehydrogenase (Lipoamide); D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000378:metabolism*
D004274:DNA, Recombinant; D010940:Plant Proteins; D011768:Pyruvate Dehydrogenase Complex; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D030481:Pyruvate Dehydrogenase (Lipoamide); C111305:pyruvate dehydrogenase E1 beta subunit; C065640:pyruvate dehydrogenase E1alpha subunit