Real-time Gene Expression Analysis as Monitoring Tool for Production of Recombinant Proteins

Abstract

In recent years numerous pharmaceuticals have been developed from biotechnologically produced proteins. In the presented study we use Escherichia coli DH5alpha with the GFP mutant gene for T-Sapphire as a model. This work describes a method to quantify gene expression of the product with real-time PCR and its potential use as a monitoring tool. Efficient total RNA purification and cDNA synthesis are one of the essential steps in real-time PCR quantification. Therefore we tested various RNA purification and reverse transcription systems for their influences on the efficiency and reproducibilityof real-time PCR. To have the possibility of comparing different experiments, normalization with housekeeping genes (HKGs) is the method of choice (relative quantification). Therefore we tested the expression of ten HKGs from various pathways for normalization. Three candidates were determined by the procedure of Vandesompele et al. 2002 (gene stability measure M, normalization factor NF). With the developed methods fermentation (batch and feed-batch) under different conditions were monitored. This work was part of the project „Monitoring and Control of Bioprocesses for heterologous protein production in Escherichia coli“ and supported by BMBF (FKZ 1710C04) running from November 2004 until January 2008.