Biochemical and Biophysical Research Communications
High-throughput expression, purification, and characterization of recombinant Caenorhabditis elegans proteins
Section snippets
Materials and methods
Expression constructs, cell culture, and lysis. The open reading frame sequences tested in this study were identified as DNA damage response (DDR) pathway interacting proteins in C. elegans that were mostly hypothetical and uncharacterized [2]. The 86 selected ORFs, which had sizes of the encoded proteins ranging from 10 to 110 kDa, were subcloned into the GATEWAY (Invitrogen) Entry vectors and subsequently transferred to the Destination vector (pDEST17) containing N-terminal 6× Histidine-tag [9]
Protein expression and solubility screening
The results of expressing 86 putative C. elegans DDR genes using the ZipTip/MALDI-MS method show that ∼62% of the genes were expressed and 5.4% of these were also soluble in the solvent tested (Fig. 2). The expressed genes were assigned as “correct mass” when the MALDI-MS measurements differ from the estimated mass based on the sequence prediction (gene plus 4 kb N- and C-terminal extra sequences) by less than 1%. The accuracy of the MALDI-MS measurements was determined by comparing to the mass
Discussion
When eukaryotic genes are expressed in prokaryotic organisms such as E. coli, they may not fold properly and may form aggregates (in the form of inclusion bodies) due to the absence of appropriate post-translational chaperones or processing [15]. However, the manipulability, short growth time, and low cost render E. coli the most widely used expression system for recombinant proteins. Therefore, many of the structural genomics projects have focused on searching for genes that express and fold
Acknowledgements
The authors thank Tim Blankenship and Elena Chernokalskaya from Millipore for their technical assistance in developing the ZipTip protein purification and Edward Nieves for his helpful suggestions on MALDI-MS methods. This research is supported in part by The Protein Structure Initiative P50-GM-62529, P41-EB-01979, and R33-CA-83179. The MALDI-MS experiments were performed in The Laboratory for Macromolecular Analysis and Proteomics (LMAP) at the Albert Einstein College of Medicine, which is
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Present address: DNA Damage Response Lab, Cancer Research UK, London Research Institute, Clare Hall, UK.