A visual method for direct selection of high-producing Pichia pastorisclones

Fan Hu,Pei Hong Mao,Jie Lü,Li Xin Ma,Xiang Jin,Peng Zheng,Xiang Dong Ma,Xin Li,Yu Lin Zhou,Sheng Yi Liu,Ben Rao,Tao Ke

doi:10.1186/1472-6750-11-23

Abstract

BackgroundThe 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.ResultsA 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.ConclusionsA 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.

Highlights

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
Recently, the methylotrophic yeast Pichia pastoris is used for high level production of recombinant proteins for basic research and medical applications [1,2,3,4]
Selection of the clones of different sizes for the mannanase activity analysis P. pastoris GS115 was transformed with linearized pHBM306 plasmid DNA and the recombinant transformants were first selected on minimal selective MD medium

Summary

Introduction

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. The methylotrophic yeast Pichia pastoris is used for high level production of recombinant proteins for basic research and medical applications [1,2,3,4]. Many heterologous proteins have been successfully expressed in Pichia pastoris due to its exceptional natural capacity for heterologous protein production http://www.kgi.edu/ Faculty-and-Research/James-M-Cregg.html. Whereas Pichia pastoris eukaryotic system has been used successfully for non-expressible proteins in E. coli [5]. Availability of new strains with mammalian-type glycosylation capabities has made P. pastoris more important for the industrial production of therapeutic proteins [8]. A good screening and selection method for high-producing Pichia pastoris clones certainly help to lower the costs significantly

Methods

Results

Discussion

Conclusion