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Over expression and purification of recombinant human myocilin

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Abstract

Human myocilin is a 55 kDa protein that is implicated in primary open angle glaucoma (POAG). Understanding the structure and folding of the native protein and the mutants that increase aggregation could lead to possible prevention of the condition. We report here the over expression and purification of the human myocilin in E. coli. The initial expression of recombinant myocilin in E. coli was found to be low. The problem of low yield was found to be due to multiple causes and was overcome using a suitable combination of vectors, tags, host background and expression protocols. The overexpressed human myocilin was purified by affinity column chromatography to yield about 8 mg of protein from 1 l of culture. The protein purity and folding were confirmed using electrophoresis, immunoblotting and fluorescence spectroscopy. Further biophysical characterization and crystallization trials using the recombinant human myocilin will pave the way for better understanding of the structure–aggregation relationship that is involved in causing POAG.

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Fig. 1
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Abbreviations

POAG:

Primary open angle glaucoma

IOP:

Intra occular pressure

TUB:

Tris urea buffer

TNB:

Tris NaCl buffer

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Acknowledgements

Department of Biotechnology, Government of India for the project support and the Bioinformatics facility at Madurai Kamaraj University (MKU). The use of facilities provided by Centre with Potential for Excellence in the subject of Genomics Sciences, Centre for Plant Molecular Biology and Genetic Engineering Research Unit at MKU. Indian Council of Medical Research, Government of India for funding. Prof V. R. Muthukaruppan for encouragement and helping to initiate the work, Dr Michael Fautsch for pRSET-Myoc. Dr J. Gowrishanker for GJ1158 strain. Dr R. Usha and Dr K. Dharmalingam for selected laboratory materials.

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Authors and Affiliations

  1. Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, 625 021, India

    P. J. Eswari Pandaranayaka & S. Krishnaswamy

  2. Department of Genetics, Aravind Medical Research Foundation, Aravind Eye Hospital, Madurai, India

    J. Kanagavalli & P. Sundaresan

  3. Glaucoma Clinic, Aravind Eye Hospital, Madurai, India

    S. R. Krishnadas

Authors
  1. P. J. Eswari Pandaranayaka
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  2. J. Kanagavalli
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  3. S. R. Krishnadas
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  4. P. Sundaresan
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  5. S. Krishnaswamy
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Corresponding author

Correspondence to S. Krishnaswamy.

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Eswari Pandaranayaka, P.J., Kanagavalli, J., Krishnadas, S.R. et al. Over expression and purification of recombinant human myocilin. World J Microbiol Biotechnol 24, 903–907 (2008). https://doi.org/10.1007/s11274-007-9549-6

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  • DOI: https://doi.org/10.1007/s11274-007-9549-6

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