Plant Molecular Biology

, Volume 11, Issue 6, pp 821–831 | Cite as

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

  • Scott Newman
  • Rose Ann Cattolico
Article

Abstract

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5′ non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.

Key words

co-transcription enzyme assembly eukaryotic algae heterologous expression Rubisco 

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Copyright information

© Kluwer Academic Publishers 1988

Authors and Affiliations

  • Scott Newman
    • 1
  • Rose Ann Cattolico
    • 1
  1. 1.Department of Botany KB-15University of WashingtonSeattleUSA
  2. 2.Zoology DepartmentDuke UniversityDurhamUSA

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