Nada Kraševec^* and Radovan Komel

National Institute of Chemistry, POB 660, SI-1001 Ljubljana, Slovenia
* Corresponding author: E-mail: nada.krasevec@ki.si;
fax: (+386)-1-4760-300

Abstract
We studied secretion of human cytokine tumour necrosis factor alpha (TNFα) by the filamentous fungus Aspergillus niger. When the gene for glucoamylase was fused with TNFα, optimized for expression in E. coli (TNFE), TNFα was detected only inside cells, in the form of an uncleaved fusion protein. Non-optimized codon usage of TNFE proved negative influence on the expression of specific mRNA. The influence of the cluster of unfavourable codons was studied further by site-directed mutagenesis in which these codons were optimized by eukaryotic codon usage, as present in human TNFα (TNFH) or in fungal glucoamylase, however, no improvement was observed. Processed TNFα was secreted only when entire TNFH replaced TNFE. If the codons of the cluster within TNFH were shifted back to bacterial codon usage, the secretion of TNFα dropped down. Due to the great impact of codon usage observed in our experiments, human cDNA seems appropriate for expression in A. niger, harbouring codons complementary enough to the fungal host specific codon usage. Methods of secondary structure prediction for mRNA proved being a helpful tool in designing gene constructs including those containing heterologous intron(s).

Keywords: Filamentous fungi; heterologous protein secretion; human tumour necrosis factor α; kexin processing; codon usage; intron.