Submitted to: Preparative Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 7, 2006
Publication Date: May 9, 2006
Citation: Miernyk, J.A., Johnston, M.L. 2006. Chemical cross-linking immobilized-concanavalin a for use in proteomic analyses. Preparative Biochemistry and Biotechnology. 36(3):203-214. Interpretive Summary: Biochemical analysis of seed development is hindered by the extreme dynamic range of the components present. The seed storage proteins make up approximately 70% of the total protein component of soybean seeds. These proteins are a transient depot for nutrients that will be used later during seed germination, and are one of the major agronomically-important components of the seeds. Thus, while biologically and economically important, the storage proteins greatly complicate biochemical analysis. A relatively simple method was developed to remove one family of seed storage proteins. However, this method was useful only after previously applying a unique chemical treatment. After this method was used on a total protein fraction from developing soybeans, it was possible to increase resolution of the non-storage protein fraction by approximately 30%. This information will be important to researchers in their attempts to increase agricultural productivity by altering the seed composition, and to other plant scientists who will try to design more efficient crop plants through either classical breeding or biotechnology.
Technical Abstract: Lectin affinity chromatography was used to reduce the amount of the abundant glycoprotein beta-conglycinin in total protein samples prepared from developing soybean (Glycine max L. Merrill cv. Jack) seeds. Electrophoretic analysis of both the concanavalin A-Sepharose binding and non-binding fraction revealed an abundant protein band at Mr 26,000. The amount of this protein was greatly increased when concanavalin A-Sepharose was used with urea-containing buffers. Peptide mass fingerprint analysis of this abundant protein band unequivocally identified it as concanavalin A (con A). A simple and gentle method was used to chemically cross-link the con A subunits so that the lectin-Sepharose retained the ability to bind high-mannose type glycoproteins. The chemically cross-linked con A-Sepharose was stable in buffers that contained up to 8M urea, making this an affinity matrix suitable for use in electrophoresis-based proteomic analyses.