Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation.
The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome.
The Structural Genomics Consortium, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom.
Tracy
T
Keates
Christopher D O
CD
Cooper
Pavel
P
Savitsky
Charles K
CK
Allerston
Claire
C
Phillips
Martin
M
Hammarström
Neha
N
Daga
Georgina
G
Berridge
Pravin
P
Mahajan
Nicola A
NA
Burgess-Brown
Susanne
S
Müller
Susanne
S
Gräslund
Opher
O
Gileadi
D000818:Animals; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002846:Chromatography, Affinity / Q000379:methods*; D003460:Crystallization; D003470:Culture Media; D005796:Genes; D006801:Humans; D013058:Mass Spectrometry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012995:Solubility
D003470:Culture Media; D020543:Proteome; D001710:Biotin