A flexible and economical medium-throughput strategy for protein production and crystallization.
Large-scale structural genomics centres rely heavily on robotics to ensure that maximum throughput is achieved. However, the size and cost of these approaches is out of the reach of most academic structural biology efforts. A major challenge for such groups is to adapt current high-throughput schemes to a reasonable scale with the resources available. A flexible medium-throughput approach has been developed that is suitable for typical academic research groups. Following nested PCR, targets are routinely cloned into two Gateway expression vectors (pDEST15 for an N-terminal GST tag and pDEST17 for an N-terminal His tag). Expression of soluble recombinant protein in Escherichia coli is rapidly assessed in 96-well format. An eight-probe sonicator is utilized and a six-buffer lysis screen was incorporated to enhance solubility. Robotics is reserved for crystallization, since this is the key bottleneck for crystallography. Screening proteins with a 480-condition protocol using a Cartesian nanolitre-dispensing robot has increased crystallization success markedly, with an overall success rate (structures solved out of proteins screened) of 19%. The methods are robust and economical -- with the exception of the crystallization robot, investment in additional equipment has been minimal at 9000 US dollars. All protocols are designed for individuals so that graduate students and postdoctoral fellows gain expertise in every aspect of the structural pipeline, from cloning to crystallization.
Moreland
Nicole
N
School of Biological Sciences, University of Auckland, Private Bag 92-019, Auckland, New Zealand. n.moreland@auckland.ac.nz
Ashton
Rachael
R
Baker
Heather M
HM
Ivanovic
Ivan
I
Patterson
Sean
S
Arcus
Vickery L
VL
Baker
Edward N
EN
Lott
J Shaun
JS
D001671:Biochemistry / Q000191:economics* / Q000379:methods*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000191:economics* / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000191:economics* / Q000379:methods*; D040901:Proteomics / Q000379:methods
D017931:DNA Primers; D046912:Multiprotein Complexes; D005982:Glutathione Transferase