The mature rat brain acetylcholinesterase gene (T subunit, AChE) was subcloned downstream of the temperature-inducible λ promoter P(L) and fused to the signal peptide of the OmpA protein. Three different expression vectors were constructed: (i) pCompmA containing the mature AChE, (ii) pCompΔTA containing a truncated AChE and (iii) pCompΔTAH containing the truncated AChE C-terminal fused to a 6xHis-tag. With all expression vectors the overexpression of AChE in Escherichia coli resulted mainly in cytoplasmic inclusion bodies (IB). However, some activity was found in the periplasmic space. The inclusion bodies were refolded in vitro, yielding up to 1.42 U/mg IB of active AChE. The refolded AChE was partially purified (approx. 300- fold) by affinity chromatography with a specific activity of approx. 250 U/mg. Removing the cysteine residue near the C-terminus (truncated AChE, ΔTAChE) assuming to affect the refolding, did not increase the amount of active enzyme obtained after refolding. Purification of denatured ΔTAChE- 6xHis prior to refolding by Ni-NTA-chromatography increased the refolding efficiency by a factor of 1.5. Functional expression and secretion of rat brain acetylcholinesterase into the medium was achieved in Pichia pastoris. By optimizing the culture conditions, 100 mU/ml AChE in the medium was produced. In this work we are describing the functional expression of a mammalian AChE in a microbial host in good yields for the first time. The physico-chemical properties of both, the bacterial and yeast expressed AChE were compared with those of the native AChE. The properties of the yeast expressed AChE and the native AChE were similar, whereas the E. coli expressed enzyme was found to be less stable and had different inhibition properties.
|Original language||English (US)|
|Number of pages||14|
|Journal||Biochimica et Biophysica Acta - Gene Structure and Expression|
|State||Published - Mar 13 1998|
Bibliographical noteFunding Information:
We are grateful to thank Prof. J. Massoulié, Laboratoire de Neurobiologie, Paris, for providing us the AChE-gene, the AChE-specific antibody and a sample of the native rat brain AChE. Dr. H. Atomi acknowledges a scholarship from the CIBA-GEIGY Foundation (Japan) for the Promotion of Sciences.
Copyright 2007 Elsevier B.V., All rights reserved.
- (E. coli)
- (Pichia pastoris)
- (Rat brain)