|Moreno, J - UNIVERSITY OF MISSOURI|
|Randall, Douglas - UNIVERSITY OF MISSOURI|
Submitted to: Protein Expression and Purification
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 15, 2000
Publication Date: March 1, 2000
Citation: MORENO, J.I., MIERNYK, J.A., RANDALL, D.D. STAPHYLOCOCCAL PROTEIN A AS A FUSION PARTNER DIRECTS SECRETION OF THE E1A AND E1B SUBUNITS OF PEA MITOCHONDRIAL PYRUVATE DEHYDROGENASE BY BACILLUS SUBTILIS. PROTEIN EXPRESSION AND PURIFICATION. 2000. V. 18(2). P. 242-248. Interpretive Summary: In layman's terms, respiration is the use of energy to do work by living cells. Both growth and reproduction are affected by respiration. As a result, respiration must be carefully controlled or wasted energy would decrease crop yields and reduce agricultural productivity. The control of respiration in plant cells is a subject of ongoing study. A complex protein that is likely to be involved in an important control step in respiration was isolated from garden pea and studied. Comparisons were made with a similar protein from animals and microbes, in order to identify which characteristics might be important in control of respiration. A method was developed to prepare large amounts of the plant protein that had full biological activity. This method will allow future studies of the gene or genes for this protein complex. Additionally, being able to produce large amounts of material will allow detailed analysis of the structures. This information will be important to researchers in their attempts to increase agricultural productivity by altering the control of plant cell respiration, and to other plant scientists who will try to design more efficient crop plants through either classical breeding or biotechnology.
Technical Abstract: Staphylococcal protein A (SPA)-based vectors were constructed to direct secretion of the E1a and E1b 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-E1a and SacBSP-E1b fusion proteins were insoluble in the cytoplasm. However, when the SPA open reading frame was inserted between SacBSP and E1a or E1b, 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- E1a and SacBSP-E-E1b). Adding the second (D) IgG binding domain improved extracellular protein yields 3-4 fold over E alone, but was not as efficient as secretion of the full-length (EDABC) SPA-fusion proteins. All constructs were based upon the pUB110-derived multicopy plasmid pWB705. Separate B. subtilis strains transformed with SacBSP-E-E1a-His6 or SacBSP- E1b were co-cultivated in the presence of Ni-NTA agarose. The native pyruvate dehydrogenase a2b2 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.