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High-throughput expression in microplate format in Pichia pastoris.
Mewes Böttner1, Christine Lang
1Berlin Technical University, Institute for Biotechnology, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|July 23, 2004
Summary
We optimized Pichia pastoris cultivation for high-throughput protein expression screening. This method efficiently identifies clones producing heterologous proteins at yields of 2 mg/L or higher.
Area of Science:
- Biotechnology
- Molecular Biology
- Microbial Engineering
Background:
- Pichia pastoris is a key host for heterologous protein expression.
- Increasing demand for diverse protein expression requires efficient screening methods.
Purpose of the Study:
- To develop an automated cultivation and induction protocol for Pichia pastoris.
- To enhance the screening of clones for high-yield heterologous protein expression.
Main Methods:
- cDNAs cloned for intracellular expression with N- and C-terminal affinity tags (6*HIS, StrepII).
- Expression controlled by the alcohol oxidase 1 (AOX1) promoter.
- Screening performed in 2 mL cultures within a 24-well format using chemical lysis.
Main Results:
- Developed an optimized feeding and induction protocol.
- Achieved identification of expression clones with protein yields of 2 mg/L or higher.
- Protocol is amenable to automation for increased clone screening capacity.
Conclusions:
- The optimized protocol significantly improves the efficiency of screening Pichia pastoris expression clones.
- This method facilitates the identification of high-producing clones for heterologous protein expression.
- The automated approach addresses the growing demand for diverse protein expression.