High-Copy Expression Vector Based on Amplification-Promoting Sequences

Abstract

We describe a new vector system that allows efficient expression of heterologous proteins in transformed mouse L fibroblasts. This is due to its persistence at high copy numbers, achieved by a 370-bp amplification promoting element (muNTS1) derived from the nontranscribed spacer of murine rDNA. Copy number determination showed that this sequence mediates a 40- to 800-fold amplification of the vector DNA in transfected L cells. High copy number was accompanied by increased expression levels of the reporter gene secreted alkaline phosphatase (SEAP). Analyzing the structural organization of multicopy plasmid DNA in mouse L cells revealed that plasmid DNA is integrated as reiterated head-to-tail concatamers into the chromosomal DNA. The vector described here can be used as a versatile high-copy expression system for heterologous proteins overcoming any limitation to enzyme-deficient cell lines.