Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs).
Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.
Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Australia.
Nikita
N
Abraham
Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Australia.
Blessy
B
Paul
Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Australia.
Lotten
L
Ragnarsson
Centre for Pain Research, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Australia.
Richard J
RJ
Lewis
D000109:Acetylcholine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008024:Ligands; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
C428944:AChBP protein, Lymnaea; D002352:Carrier Proteins; D008024:Ligands; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D000109:Acetylcholine