Influence of Primary Gene Structure on Secretion of Recombinant TNF ALFA From Aspergillus niger

We studied secretion of human cytokine tumour necrosis factor alpha (TNF alfa) by the filamentous fungus Aspergillus niger. When the gene for glucoamylase was fused with TNFalfa, optimized for expression in E. coli (TNFE), TNFalfawas 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 TNFalfa(TNFH) or in fungal glucoamylase, however, no improvement was observed. Processed TNFalfawas 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 TNFalfa 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).