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Purification, cloning and expression of spinach leaf sucrose-phosphate synthase in Escherichia coli by U. Sonnewald; W. P. Quick; E. MacRae; K.-P. Krause; Mark Stitt is a Agricultural and Biological Sciences article available to read on EtoBox.

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Sucrose-phosphate synthase (SPS) from leaves of spinach (Spinacia oleracea L.) has been purified to homogeneity by a procedure involving precipitation with polyethylenglycol and chromatography over diethylaminoethylcellulose, f~-aminohexylagarose, Mono Q and Blue Affinity columns. The purification factor was 838 and the final specific activity was 1.3 nkat.(mg protein)-1. On denaturing gels the major polypeptide was 120 kDa but there was also a variable amount of smaller polypeptides in the range of 90 to 110 kDa. A new activity stain was developed to allow visualization of SPS in gels. The holoenzyme had a molecular weight of about 240 and 480 kDa in native gels and Sepharose, respectively. A high-titre polyclonal antibody was obtained which reacted with SPS from other species including wheat, potato, banana and maize. Screening of a spinach-leaf cDNA-expression library with the antibody allowed the isolation of a full-length clone. Sequencing revealed a predicted molecular weight of 117649 Da, and considerable homology with the recently published sequence for maize leaf (Worrell et al. 1991, Plant Cell 3, 1121-1130). Expression of the spinach-leaf SPS gene in Escherichia coli res

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Author

U. Sonnewald; W. P. Quick; E. MacRae; K.-P. Krause; Mark Stitt

Publisher

Springer-Verlag; Springer Verlag; Springer Science and Business Media LLC; Society for Mining, Metallurgy and Exploration Inc. (ISSN 0032-0935)

Published

1993

Language

EN

Field

Agricultural and Biological Sciences (Life Sciences)