PMID: 9545588Apr 18, 1998Paper

Functional expression of a mammalian acetylcholinesterase in Pichia pastoris: comparison to acetylcholinesterase, expressed and reconstituted from Escherichia coli

Biochimica Et Biophysica Acta
J HeimRolf D Schmid

Abstract

The mature rat brain acetylcholinesterase gene (T subunit, AChE) was subcloned downstream of the temperature-inducible lambda promoter PL and fused to the signal peptide of the OmpA protein. Three different expression vectors were constructed: (i) pCompmA containing the mature AChE, (ii) pComp delta TA containing a truncated AChE and (iii) pComp delta TAH 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, delta TAChE) assuming to affect the refolding, did not increase the amount of active enzyme obtained after refolding. Purification of denatured delta TAChE-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 wa...Continue Reading

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Citations

Jan 21, 2000·FEMS Microbiology Reviews·J L Cereghino, J M Cregg
Dec 18, 2002·Biosensors & Bioelectronics·Holger SchulzeRolf D Schmid
Sep 7, 2000·Experimental Eye Research·J Jimenez-Asensio, D Garland
May 16, 2017·Journal of Neurochemistry·Israel Silman, Joel L Sussman
Oct 9, 2001·Biotechnology and Bioengineering·T S MorH S Mason
Jan 28, 2005·The Tohoku Journal of Experimental Medicine·Osamu HorieRyukichi Ryo

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