Genome-scale protein expression and structural biology of Plasmodium falciparum and related Apicomplexan organisms.
Parasites from the protozoan phylum Apicomplexa are responsible for diseases, such as malaria, toxoplasmosis and cryptosporidiosis, all of which have significantly higher rates of mortality and morbidity in economically underdeveloped regions of the world. Advances in vaccine development and drug discovery are urgently needed to control these diseases and can be facilitated by production of purified recombinant proteins from Apicomplexan genomes and determination of their 3D structures. To date, both heterologous expression and crystallization of Apicomplexan proteins have seen only limited success. In an effort to explore the effectiveness of producing and crystallizing proteins on a genome-scale using a standardized methodology, over 400 distinct Plasmodium falciparum target genes were chosen representing different cellular classes, along with select orthologues from four other Plasmodium species as well as Cryptosporidium parvum and Toxoplasma gondii. From a total of 1008 genes from the seven genomes, 304 (30.2%) produced purified soluble proteins and 97 (9.6%) crystallized, culminating in 36 crystal structures. These results demonstrate that, contrary to previous findings, a standardized platform using Escherichia coli can be effective for genome-scale production and crystallography of Apicomplexan proteins. Predictably, orthologous proteins from different Apicomplexan genomes behaved differently in expression, purification and crystallization, although the overall success rates of Plasmodium orthologues do not differ significantly. Their differences were effectively exploited to elevate the overall productivity to levels comparable to the most successful ongoing structural genomics projects: 229 of the 468 target genes produced purified soluble protein from one or more organisms, with 80 and 32 of the purified targets, respectively, leading to crystals and ultimately structures from one or more orthologues.
Vedadi
Masoud
M
Structural Genomics Consortium, U. of Toronto, 100 College St. Rm 522B, Toronto, Ont., Canada M5G 1L5.
Lew
Jocelyne
J
Artz
Jennifer
J
Amani
Mehrnaz
M
Zhao
Yong
Y
Dong
Aiping
A
Wasney
Gregory A
GA
Gao
Mian
M
Hills
Tanya
T
Brokx
Stephen
S
Qiu
Wei
W
Sharma
Sujata
S
Diassiti
Angelina
A
Alam
Zahoor
Z
Melone
Michelle
M
Mulichak
Anne
A
Wernimont
Amy
A
Bray
James
J
Loppnau
Peter
P
Plotnikova
Olga
O
Newberry
Kate
K
Sundararajan
Emayavaram
E
Houston
Simon
S
Walker
John
J
Tempel
Wolfram
W
Bochkarev
Alexey
A
Kozieradzki
Ivona
I
Edwards
Aled
A
Arrowsmith
Cheryl
C
Roos
David
D
Kain
Kevin
K
Hui
Raymond
R
D000818:Animals; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018503:Genome, Protozoan / Q000235:genetics*; D008958:Models, Molecular; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
D015800:Protozoan Proteins