The bacterial expression, in vitro refolding, proteolytic processing and preliminary crystallization of the 11S globulin subunit, legumin B4, from faba bean (Vicia faba var. Minor)
Abstract
The 11S and 7S globulins are major seed storage proteins in agriculturally important legumes such as faba bean (Vicia faba) and soybean (Glycine max), and serve as a source of protein for animals and mankind. Unfortunately, these proteins are low in sulfur-containing amino acids. Attempts to alter the amino acid content of the seed by gene modification and their reintroduction into the plant have failed to produce transgenic plants that express the mutant protein. Changes to the 11S storage protein described thus far have resulted in proteins that are degraded and not accumulated. Understanding the three-dimensional structure of the globulins may aid in predictions about which amino acids could be changed without affecting the structure of the protein. Although the three-dimensional structure is known for two 7S globulins, phaseolin and canavalin, the three-dimensional structure of the 11S globulin remains unsolved. This thesis describes the construction of a recombinant 11S globulin comprised entirely of legumin B4 subunits. Also described are the bacterial expression conditions for three 11S subunits and their assembly into prolegumin trimers equivalent to those found in the endoplasmic reticulum as determined by sucrose density gradient centrifugation. The legumin B4 trimers were processed by an asparaginyl endopeptidase, a step necessary for further assembly of molecules into the mature 11S globulin hexamers. The hexamers are equivalent to those isolated from protein storage vacuoles of seeds. Immature proglobulin trimer and mature 11S globulin hexamers were used for preliminary crystallization experiments. The 7S proglobulin trimers crystallized as a single crystal morphology, rhombohedral needles. These crystals were too disordered to give an x-ray diffraction pattern. The 11S globulin crystallized as three different morphologies: rhombohedral, hexagonal, and three-pointed star crystals. These crystals were too small to put in an x-ray beam, however they provide a good starting point for future crystallization experiments.
Degree
Ph.D.
Advisors
Nielsen, Purdue University.
Subject Area
Agronomy|Food science|Biochemistry
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