Expression of human proteins at the Southeast Collaboratory for Structural Genomics.
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.
Mayer
Michael R
MR
Southeast Collaboratory for Structural Genomics, A222 Life Sciences, Athens, GA 30602-7229, USA. mmayer@secsg.org
Dailey
Tamara A
TA
Baucom
Clay M
CM
Supernak
Jill L
JL
Grady
Michael C
MC
Hawk
Harris E
HE
Dailey
Harry A
HA
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
D011994:Recombinant Proteins