Influence of charge variation in the Streptomyces venezuelae a-amylase signal peptide on heterologous protein production by Streptomyces lividans

Abstract

With the aim of investigating determining factors for secretion of heterologous proteins by streptomycetes, we analysed the effect of charge variation in the Streptomyces venezuelae ATCC15068 α-amylase signal peptide on expression and secretion of mouse tumour necrosis factor α (mTNF) by Streptomyces lividans. To this end, the mTNF cDNA was fused to the wild-type α-amylase (aml) signal sequence and the fusion gene was expressed under the control of the S. venezuelae CBS762.70 subtilisin inhibitor gene (vsi) promoter, which has been shown to be very effective in initiating transcription. In addition, the number of positive charges in the N region of the α-amylase signal peptide was altered by in vitro mutagenesis. Secreted and intracellular mTNF levels were determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and biologi-cal activity measurements. This revealed moderate amounts of secreted mTNF compared to the levels obtained in previous experiments using the vsi promoter in combination with the Vsi signal peptide. Levels of secreted mTNF could be increased sevenfold by introducing one extra positive charge in the N region of the signal peptide.