Expression system of Pyrus pyrifolia S-RNase in E. coli and its application to screening of pollen S-gene product(s)

Gamage. N., Matsuura. T., Norioka, N., Yoshimura. Y., Nakanishi, T., and Norioka. S.

Division of Protein Chemistry, Institute for Protein Research, Osaka University, Japan

International Conference on Pollen-Stigma Interactions (Oxford, United Kingdom, 1999.7.18-21)

Rosaceous plants, such as pear and apple, show gametophytic self-incompatibility controlled by a single locus (S-Locus) with multiple alleles (S-alleles), which encodes a ribonuclease expressed exclusively in the styles (S-RNase). In Pyrus pyrifolia Japanese pear, seven S-alleles (Sl~S7) are known and their gene products in pistil (Sl~S7-RNase) have been identified. However, the mechanism of the self-incompatibility in Rosaceae still remains unclear because pollen S-gene product(s), which interact with the S-RNases and responsible for self and non-self recognition between pollen and pistil, has not been found yet. To search for pol]en S-gene product(s) by affinity chromatography we constructed an expression system of glutathione-S-transferase (GST) and S-RNase fusion protein (GST-S-RNase) in E. coli. The cDNA encoding S3-RNase was inserted into the multicloning site of pGEX-4T-3 vector which enables proteins to be expressed as a fusion protein with GST. E. coli carrying this expression vector was grown at 20 °C to midlogarithmic phase and expression of the GST-3-RNase was induced by addition of IPTG (0.5 mM). The cells were harvested and lysed. After centrifugation, subernatant was applied to a glutathione-Sepharose column to.purify GST-S3-RNase. The SDS-PAGE and western blot analyses of the eluate with 10 mM glutathione from the column revealed that the GST-S3-RNase was expressed in E. coli as a complex with chaperones, GroEL and DnaK. These chaperones were dissociated from the GST-S3-RNase by incubation with 10 mM Mg-ATP and we could isolate the fusion protein. This fusion protein is useful for screening of pollen S-gene product(s) as a ligand of affinity chromatography.

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MATSUURA Takanori (Please change the mark “%” to “@”)

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