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Open Access Highly Accessed Methodology article

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

Fan Hu1, Xin Li2, Jie Lü3, Pei Hong Mao3, Xiang Jin3, Ben Rao1, Peng Zheng1, Yu Lin Zhou1, Sheng Yi Liu2, Tao Ke4, Xiang Dong Ma1* and Li Xin Ma1

Author Affiliations

1 Hubei Key Laboratory of Industrial Biotechnology, College of Life Science, Hubei University, Wuhan, 430062, PR China

2 Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, 430062, PR China

3 Institute of Ion Beam Biotechnology, College of Physics Science and Technology, Xinjiang University, Urumqi, 830008, PR China

4 College of Life Science and Technology, Nanyang Normal University, Nanyang 473061, PR China

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BMC Biotechnology 2011, 11:23  doi:10.1186/1472-6750-11-23

Published: 21 March 2011

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.