title: Expression of human proteins at the Southeast Collaboratory for Structural Genomics.
abstract: The human protein production group at the Southeast Collaboratory for Structural Genomics is charged with producing human proteins for both X-ray crystallography and NMR structural studies. Eukaryotic, and human proteins in particular, are notoriously difficult to express in bacterial systems. For various reasons, T7-based expression often results in protein expressed in an insoluble form. Overcoming this requires either introduction of a step to screen expression conditions or inclusion of a troublesome refolding step during purification. Our laboratory uses a trc-based expression vector that addresses many of the difficulties of the commonly used T7-based expression systems. Proteins expressed under the trc promoter, a weak promoter compared to the strong T7 promoter, are produced in a soluble form and include necessary cofactors. The details of this system will be discussed.
authors.affiliation: Southeast Collaboratory for Structural Genomics, A222 Life Sciences, Athens, GA 30602-7229, USA. mmayer@secsg.org
authors.forename: Michael R
authors.initials: MR
authors.lastname: Mayer
authors.forename: Tamara A
authors.initials: TA
authors.lastname: Dailey
authors.forename: Clay M
authors.initials: CM
authors.lastname: Baucom
authors.forename: Jill L
authors.initials: JL
authors.lastname: Supernak
authors.forename: Michael C
authors.initials: MC
authors.lastname: Grady
authors.forename: Harris E
authors.initials: HE
authors.lastname: Hawk
authors.forename: Harry A
authors.initials: HA
authors.lastname: Dailey
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods*; D005845:Georgia; D006801:Humans; D007753:Laboratories; D015394:Molecular Structure; D011401:Promoter Regions, Genetic; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D012995:Solubility
chemical_list: D011994:Recombinant Proteins