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Abstract. Proso millet glutelin protein fractions make up its second-largest protein mostly used in proso millet concentrates and isolates. In this experiment, we identified the gene of glutelin type-B 5-like protein isoform of the glutelin fraction with over 40% structural identity with the crystal structure of recombinant pro-11S globulin of pumpkin (2E9Q). We further cloned the gene of glutelin type-B 5-like protein with attached six continuous histidine codons into a pET 21d expression vector, overexpressed in rosetta Escherichia coli cells, purified the protein from inclusion bodies by unfolding with 8 M urea and refolding in 0.5 M Arginine with 20 mM CHES (2-(Cyclohexylamino)ethanesulfonic acid). Characterization of purified protein reveals above 90% pure protein based on an SDS-PAGE gel analysis with a band 22.1 kDa. Additionally, the protein revealed a monodispersed particle size distribution with a broad range of particle size (6.50 – 43.82 nm) and a higher-order aggregation with molecular weight range 60-70 kDa.