Recombinant Human Acetylcholinesterase - Production and Post-Translation Processing.
Abstract
Genetic engineering techniques were employed for development of efficient expression systems of recombinant human acetylcholinesterase (rHuAChE). Human cell lines allowing production of 10-20 mg/liter of fully-active enzyme were established. Biotechnological processes for large scale production of rHuAChE from these cells were evaluated in multitray systems, fixed-bed reactors and microcarrier cultures. The latter proved to be the most promising, exhibiting system productivity of 6-10 mg/liter/day. rHuAChE is secreted from the recombinant cells in the soluble homo-oligomeric form. Analysis of post-translation processing events indicate that the nascent molecules acquire 2-3 sugar side chains and that dimerization via disulfide bonds occurs early within the endoplasmic reticulum. The signal sequences for these modifications were identified by site-directed mutagenesis: Asn-265, Asn-350 and Asn-464 appear to be targets for N-glycosylation, Cys-580 is involved in dimerization of subunits, whereas other residues at the C-terminus may control rates of intracellular transport and assembly. Mutagenesis at all these sites did not interfere with the catalytic activity. Such mutations could be utilized, in the future, for engineering of rHuAChE molecules with altered physicochemical and pharmacological characteristics.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 13, 1993
- Accession Number
- ADP008864
Entities
People
- A. Lazar
- A. Ordentlich
- B. Velan
- C. Kronman
- Y. Flashner
Organizations
- Israel Institute for Biological Research