Characterization and intracellular distribution of pea phytochrome I Polypeptides expressed in E. coli

The pea phytochrome I (PI) cDNA clone, pPP1001, was expressed in E. coli. The plasmid pPP1001 contains pea PI cDNA which covers the entire coding region with the Shine-Dalgarno consensus sequence joined upstream of the cDNA in an expression vector pNUT6. The pPP1001 transformants formed typical inclusion bodies when cultured at 32°C. However, when cultured at 37°C or in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG) at 32°C, the bacteria lysed before inclusion body formation. Immuno-staining with anti-PI monoclonal antibody, mAP5, of transformants fixed by cold methanol showed that stainable materials were distributed in whole cytoplasmic region. When the inclusion bodies were observed clearly, the regions corresponding to the inclusion bodies became difficult to stain. Western blot analysis, however, showed that a ca. 100 kDa PI polypeptide was detected in the fraction from inclusion bodies and a ca. 90 kDa PI polypeptide from the soluble fraction. The amino acid sequence analysis of purified 100 kDa PI sample indicated that its amino terminus is blocked. However, minor signals in one experiment yielded a sequence corresponding to the expected amino terminus of pea PI except for the initiation methionine. One of the anti-pea PI monoclonal antibodies, mAP9, that recognizes the near N-terminus of pea phytochrome was reactive to the 100 kDa polypeptide.