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Constitutive Expression and Optimization of Nutrients for Streptokinase Production by Pichia pastoris Using Statistical Methods

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Abstract

The Pichia pastoris clone producing streptokinase (SK) was optimized for its nutritional requirements to improve intracellular expression using statistical experimental designs and response surface methodology. The skc gene was ligated downstream of the native glyceraldehyde 3-phosphate dehydrogenase promoter and cloned in P. pastoris. Toxicity to the host was not observed by SK expression using YPD medium. The transformant producing SK at level of 1,120 IU/ml was selected, and the medium composition was investigated with the aim of achieving high expression levels. The effect of various carbon and nitrogen sources on SK production was tested by using Plackett–Burman statistical design and it was found that dextrose and peptone are the effective carbon and nitrogen sources among all the tested. The optimum conditions of selected production medium parameters were predicted using response surface methodology and the maximum predicted SK production of 2,136.23 IU/ml could be achieved with the production medium conditions of dextrose (x1), 2.90%; peptone (x2), 2.49%; pH, 7.2 (x3), and temperature, 30.4 (x4). Validation studies showed a 95% increase in SK production as compared to that before optimization at 2,089 IU/ml. SK produced by constitutive expression was found to be functionally active by plasminogen activation assay and fibrin clot lysis assay. The current recombinant expression system and medium composition may enable maximum production of recombinant streptokinase at bioreactor level.

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Acknowledgements

This study was supported by a funding under the Technology Education Quality Improvement Program (TEQIP) by World Bank to Centre for Biotechnology, JNTU, Hyderabad. RNV acknowledges Council of Scientific and Industrial Research (CSIR) India for research fellowship.

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Authors and Affiliations

  1. Centre for Biotechnology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad, 500085, India

    Ravi Nagaraj Vellanki & Lakshmi Narasu Mangamoori

  2. Bioengineering and Environmental Centre, Indian Institute of Chemical Technology (CSIR), Tarnaka, Hyderabad, 500607, India

    Ravichandra Potumarthi

  3. Centre for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad, 500085, India

    Lakshmi Narasu Mangamoori

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  1. Ravi Nagaraj Vellanki
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  2. Ravichandra Potumarthi
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  3. Lakshmi Narasu Mangamoori
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Correspondence to Lakshmi Narasu Mangamoori.

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Vellanki, R.N., Potumarthi, R. & Mangamoori, L.N. Constitutive Expression and Optimization of Nutrients for Streptokinase Production by Pichia pastoris Using Statistical Methods. Appl Biochem Biotechnol 158, 25–40 (2009). https://doi.org/10.1007/s12010-008-8315-z

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  • DOI: https://doi.org/10.1007/s12010-008-8315-z

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