title: High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease.
abstract: The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 proteins per year at a rate of eight purifications per week. The pipeline was implemented using two ÄKTAexplorer 100 s and four ÄKTAprimes to perform immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. Purifications were completed in a period of 5 d and yielded an average of 53 mg highly purified protein. This paper provides a detailed description of the methods used to purify, characterize and store SSGCID proteins. Some of the purified proteins were treated with 3C protease, which was expressed and purified by UW-PPG using a similar protocol, to cleave non-native six-histidine tags. The cleavage was successful in 94% of 214 attempts. Cleaved proteins yielded 2.9% more structures than uncleaved six-histidine-tagged proteins. This 2.9% improvement may seem small, but over the course of the project the structure output from UW-PPG is thus predicted to increase from 260 structures to 318 structures. Therefore, the outlined protocol with 3C cleavage and subtractive IMAC has been shown to be a highly efficient method for the standardized purification of recombinant proteins for structure determination via X-ray crystallography.
authors: Seattle Structural Genomics Center for Infectious Disease (SSGCID), USA.
authors: Cassie M
authors: CM
authors: Bryan
authors: Janhavi
authors: J
authors: Bhandari
authors: Alberto J
authors: AJ
authors: Napuli
authors: David J
authors: DJ
authors: Leibly
authors: Ryan
authors: R
authors: Choi
authors: Angela
authors: A
authors: Kelley
authors: Wesley C
authors: WC
authors: Van Voorhis
authors: Thomas E
authors: TE
authors: Edwards
authors: Lance J
authors: LJ
authors: Stewart
mesh_terms: D003141:Communicable Diseases; D023281:Genomics; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011506:Proteins