Studies on The Production of Single-chain Antibody Fragments by Aspergillus Species

Hanny (J.)G..M. Hessing1, Leon G.J. Frenken2 Marian van Muijlwijk-Hartveld1, Wouter Musters2 and Cees A.M.J.J.

Author address: 

van den Hondel1. 1Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research,

Abstract: 

Filamentous fungi, such as Aspergillus niger and Aspergillus awamori, are able to produce extracellularly significant amounts of homologous proteins. Furthermore, they have the capacity to secrete heterologous proteins although to a much lesser extent. We investigated their capacity for production of single-chain antibody (scFv-) fragments. These fragments comprise the variable fragment of an antibody heavy chain connected via a linker peptide to a variable fragment of a light chain. We studied a.o. the production of scFv-anti Hen egg white lysozyme [scFv-LYS; (1)] as a model. For expression of the scFv encoding sequence expression cassettes were made in which the gene to be expressed was flanked by efficient expression signals such as the A.niger glucoamylase promoter (2) or signals derived from the A.awamori 1,4-B-endoxylanase A gene (3). Transformants comprising multiple copies of the expression cassette produced up to 10 mgA scFvLYS in the presence of a signal sequence. However, when scFv-LYS was expressed as a fusion protein with glucoamylase - a protein which is well secreted by A. niger- production levels increased at least five-fold. To obtain mature scFv-LYS, a KEX2 cleavage site was inserted between the glucoamylase and the scF-v-1,YS sequences. More data on the production of scFv-LYS and on the production of other scFv-antibody fragments will be presented. 1. Ward S. et al; Nature 341 (1989) 544-546; 2. Verdoes J.C et al; J. of Biotechnology 36 (1994) 165-175 3. J.G.M. Hessing et al,- Curr. Genet. 26 (1994) 228-232
1996

abstract No: 

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Full conference title: 

3rd EUROPEAN CONFERENCE ON FUNGAL GENETICS
    • ECFG 3rd (1996)