A visual method for direct selection of high-producing <it>Pichia pastoris </it>clones

• Main Authors: Liu Sheng, Zhou Yu, Zheng Peng, Rao Ben, Jin Xiang, Mao Pei, Lü Jie, Li Xin, Hu Fan, Ke Tao, Ma Xiang, Ma Li
• Format: Article
• Language: English
• Published: BMC 2011-03-01
• Series: BMC Biotechnology
• Online Access: http://www.biomedcentral.com/1472-6750/11/23

<p>Abstract</p> <p>Background</p> <p>The methylotrophic yeast, <it>Pichia pastoris</it>, 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, <it>P. pastoris </it>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.</p> <p>Results</p> <p>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 <it>Pichia pastoris </it>using this technology.</p> <p>Conclusions</p> <p>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 <it>Saccharomyces cerevisiae </it>for the selection of clones.</p>