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Generation and characterization of His-tagged-PsbA-expressing transformants of Chlamydomonas reinhardtii that are capable of photoautotrophic growth

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Abstract

Histidine tags attached to subunits of photosystem II (PSII) have proven to be very useful tools for isolation and purification of the complex for investigation of its components. However, it has been reported that Chlamydomonas reinhardtii transformants carrying N-terminal histidine-tagged version of the PSII D1 reaction center protein could not grow photoautotrophically. We report here a successful generation of C. reinhardtii transformants expressing histidine-tagged version of the PsbA protein that are capable of photoautotrophic growth. Biochemical and physiological analyses revealed that the histidine tag present at the N terminus of the D1 subunit did not cause total instability to the PSII complex as assessed by their phototrophic growth and oxygen evolution capability. Simple one-step affinity column chromatography also revealed that the histidine-tagged D1 subunit as well as its associated proteins could be effectively purified. These transformants could potentially serve as very good tools for the study of the PSII complex particularly the D1 protein.

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Acknowledgments

This work was supported by funding, in part, from Mahidol University, The Office of the Higher Education Commission, and Thailand Research Fund. TJ was a graduate student supported by the Royal Golden Jubilee Ph.D. Program. The authors thank Prof Anastasios Melis for providing the polyclonal antibodies against the D1 and LHC-II proteins and Dr Sarawut Jitrapakdee for monoclonal antibody specific to 6× histidine tag. We also thank Dr Jun Minagawa for providing the plasmid pBAH158.

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Authors and Affiliations

  1. Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, 272 Rama 6 Rd, Bangkok, 10400, Thailand

    Janewit Wongratana, Chutima Sereeruk & Kittisak Yokthongwattana

  2. Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhon Pathom, 73170, Thailand

    Thanate Juntadech & Chanan Angsuthanasombat

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  1. Janewit Wongratana
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  2. Thanate Juntadech
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  3. Chutima Sereeruk
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  4. Chanan Angsuthanasombat
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  5. Kittisak Yokthongwattana
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Correspondence to Kittisak Yokthongwattana.

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Wongratana, J., Juntadech, T., Sereeruk, C. et al. Generation and characterization of His-tagged-PsbA-expressing transformants of Chlamydomonas reinhardtii that are capable of photoautotrophic growth. J Appl Phycol 25, 445–452 (2013). https://doi.org/10.1007/s10811-012-9878-1

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  • DOI: https://doi.org/10.1007/s10811-012-9878-1

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