Segregational Instability in E. Coli of Expression Plasmids Carrying Human Interferon Gamma Gene and its 3’-End Truncated Variants

ABSTRACT

Segregation of the expression plasmids under non-selective conditions is due to the irregular distribution of the plasmid molecules between the daughter cells during cell division. Thus a heterogeneous population of cells appears in which the non-productive plasmid-free cells overgrow the plasmid-bearing once. The main factors that influence plasmid segregational instability are: plasmid structure, plasmid copy number, host cell genotype, level of protein expression, fermentation conditions, etc. The aim of this work is to investigate the effect of recombinant gene structure and level of expression on the segregation of expression plasmids based on pBR322. To this end the wild type human interferon gamma (hIFNγ) gene and a series of seven derivative genes obtained by systemic 3′-end deletion were inserted into an expression plasmid under a strong constitutive promoter. The segregational instability was studied under batch fermentation conditions in non-selective Luria Bertani medium, where the cell cultures were maintained at an exponential growth phase. To describe the population dynamics, a mathematical model proposed by Stewart & Levin was used and the parameters Δ (difference in specific growth rate between plasmid-free and plasmid-harbouring cells) and θ (relative plasmid loss rate) were determined for each plasmid. The obtained results showed that any variation in the 3′-end of the hIFNγ-gene strongly affected plasmid instability. We also found that the relative plasmid loss rate θ was proportional to the hIFNγ-mRNA content in the cell. Based on these results, we conclude that the structure of the constitutively expressed gene is of great importance for the segregational plasmid instability.

Keywords:

• Escherichia coli
• human interferon gamma
• segregational plasmid instability