Automated Purification of Recombinant Proteins: Combining High-throughput with High Yield

Journal Article · Mon May 01 04:00:00 EDT 2006 · Protein expression and purification, 47(1):16-24

DOI:https://doi.org/10.1016/j.pep.2005.11.015· OSTI ID:885443

Lin, Chiann Tso; Moore, Priscilla A; Auberry, Deanna L; Landorf, Elizabeth V; Peppler, Teresa; Victry, Kristin D; Collart, Frank R; Kery, Vladimir

Protein crystallography, mapping protein interactions and other approaches of current functional genomics require not only purifying large numbers of proteins but also obtaining sufficient yield and homogeneity for downstream high-throughput applications. There is a need for the development of robust automated high-throughput protein expression and purification processes to meet these requirements. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli: First - a filtration separation protocol based on expression of 800 ml E. coli cultures followed by filtration purification using Ni2+-NTATM Agarose (Qiagen). Second - a smaller scale magnetic separation method based on expression in 25 ml cultures of E.coli followed by 96-well purification on MagneHisTM Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins about 8 ug of purified protein per unit of OD at 600 nm of bacterial culture. We discuss advantages and limitations of the automated workflows that can provide proteins more than 90 % pure in the range of 100 ug – 45 mg per purification run as well as strategies for optimization of these protocols.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 885443
• Report Number(s): PNNL-SA-45902; 6504; KP1501021
• Journal Information: Protein expression and purification, 47(1):16-24, Journal Name: Protein expression and purification, 47(1):16-24 Journal Issue: 1 Vol. 47
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CRYSTALLOGRAPHY
ESCHERICHIA COLI
Environmental Molecular Sciences Laboratory
FILTRATION
FUNCTIONALS
GENES
High throughput
automated
protein expression
protein purification
filtration
magnetic separation
OPTIMIZATION
PROTEINS
PURIFICATION