原创力文档a visual method for direct selection of high-producing pichia pastoris clones直接选择的视觉方法高产毕赤酵母属pastoris克隆.pdfVIP
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a visual method for direct selection of high-producing pichia pastoris clones直接选择的视觉方法高产毕赤酵母属pastoris克隆
Hu et al. BMC Biotechnology 2011, 11:23
/1472-6750/11/23
METHODOLOGY ARTICLE Open Access
A visual method for direct selection of
high-producing Pichia pastoris clones
1† 2† 3 3 3 1 1 1 2
Fan Hu , Xin Li , Jie Lü , Pei Hong Mao , Xiang Jin , Ben Rao , Peng Zheng , Yu Lin Zhou , Sheng Yi Liu ,
Tao Ke4, Xiang Dong Ma1*, Li Xin Ma1
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.
Background coli might produce a misfolded protein that is usually
Recently, t
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