Automated, high-throughput platform for protein solubility screening using a split-GFP system |
| |
Authors: | Pawel Listwan Thomas C Terwilliger Geoffrey S Waldo |
| |
Institution: | (1) Bioscience Division, MS-M888, Los Alamos National Laboratory, Bikini Atoll Rd, SM30, Los Alamos, NM 87545, USA |
| |
Abstract: | Overproduction of soluble and stable proteins for functional and structural studies is a major bottleneck for structural genomics
programs and traditional biochemistry laboratories. Many high-payoff proteins that are important in various biological processes
are “difficult to handle” as protein reagents in their native form. We have recently made several advances in enabling biochemical
technologies for improving protein stability (), allowing stratagems for efficient protein domain trapping, solubility-improving mutations, and finding protein folding
partners. In particular split-GFP protein tags are a very powerful tool for detection of stable protein domains. Soluble,
stable proteins tagged with the 15 amino acid GFP fragment (amino acids 216–228) can be detected in vivo and in vitro using
the engineered GFP 1–10 “detector” fragment (amino acids 1–215). If the small tag is accessible, the detector fragment spontaneously
binds resulting in fluorescence. Here, we describe our current and on-going efforts to move this process from the bench (manual
sample manipulation) to an automated, high-throughput, liquid-handling platform. We discuss optimization and validation of
bacterial culture growth, lysis protocols, protein extraction, and assays of soluble and insoluble protein in multiple 96
well plate format. The optimized liquid-handling protocol can be used for rapid determination of the optimal, compact domains
from single ORFS, collections of ORFS, or cDNA libraries. |
| |
Keywords: | Automation High-throughput screening Green fluorescent protein Protein expression Protein solubility Robotics |
本文献已被 SpringerLink 等数据库收录! |
|