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A visual method for direct selection of high-producing Pichia pastoris clones

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Author(s): Hu Fan | Li Xin | Lü Jie | Mao Pei | Jin Xiang | Rao Ben | Zheng Peng | Zhou Yu | Liu Sheng | Ke Tao | Ma Xiang | Ma Li

Journal: BMC Biotechnology
ISSN 1472-6750

Volume: 11;
Issue: 1;
Start page: 23;
Date: 2011;
Original page

ABSTRACT
Abstract Background The methylotrophic yeast, Pichia pastoris, offers the possibility to generate a high amount of recombinant proteins in a fast and easy way to use expression system. Being a single-celled microorganism, P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. A simple and direct method for the selection of high-producing clones can dramatically enhance the whole production process along with significant decrease in production costs. Results A visual method for rapid selection of high-producing clones based on mannanase reporter system was developed. The study explained that it was possible to use mannanase activity as a measure of the expression level of the protein of interest. High-producing target protein clones were directly selected based on the size of hydrolysis holes in the selected plate. As an example, the target gene (9elp-hal18) was expressed and purified in Pichia pastoris using this technology. Conclusions A novel methodology is proposed for obtaining the high-producing clones of proteins of interest, based on the mannanase reporter system. This system may be adapted to other microorganisms, such as Saccharomyces cerevisiae for the selection of clones.
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