title: Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes.
abstract: As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.
authors.affiliation: Structural Genomics of Pathogenic Protozoa, Box 357350, University of Washington, Seattle, WA 98195, USA. cmehlin@u.washington.edu
authors.forename: Christopher
authors.initials: C
authors.lastname: Mehlin
authors.forename: Erica
authors.initials: E
authors.lastname: Boni
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Buckner
authors.forename: Linnea
authors.initials: L
authors.lastname: Engel
authors.forename: Tiffany
authors.initials: T
authors.lastname: Feist
authors.forename: Michael H
authors.initials: MH
authors.lastname: Gelb
authors.forename: Lutfiyah
authors.initials: L
authors.lastname: Haji
authors.forename: David
authors.initials: D
authors.lastname: Kim
authors.forename: Colleen
authors.initials: C
authors.lastname: Liu
authors.forename: Natascha
authors.initials: N
authors.lastname: Mueller
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Myler
authors.forename: J T
authors.initials: JT
authors.lastname: Reddy
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Sampson
authors.forename: E
authors.initials: E
authors.lastname: Subramanian
authors.forename: Wesley C
authors.initials: WC
authors.lastname: Van Voorhis
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Worthey
authors.forename: Frank
authors.initials: F
authors.lastname: Zucker
authors.forename: Wim G J
authors.initials: WG
authors.lastname: Hol
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007526:Isoelectric Point; D016366:Open Reading Frames; D010963:Plasmodium falciparum / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins