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- Title
A visual method for direct selection of high-producing Pichia pastoris clones.
- Authors
Fan Hu; Xin Li; Jie Lü; Pei Hong Mao; Xiang Jin; Ben Rao; Peng Zheng; Yu Lin Zhou; Sheng Yi Liu; Tao Ke; Xiang Dong Ma; Li Xin Ma
- 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.
- Subjects
YEAST; PICHIA pastoris; METHYLOTROPHIC microorganisms; RECOMBINANT proteins; MOLECULAR cloning
- Publication
BMC Biotechnology, 2011, Vol 11, Issue 1, p23
- ISSN
1472-6750
- Publication type
Article
- DOI
10.1186/1472-6750-11-23