Identification of an attenuating region in the bovine follicle-stimulating hormone beta subunit mRNA that decreases its expression in E. coli.

Abstract

Follicle-stimulating hormone (FSH) is one of the key regulators of gonadal function in mammals. Recombinant DNA expression of this hormone has proved to be a difficult task as expression levels are invariably low, irrespective of the expression system employed. In the present study, we have attempted to identify reasons for this low expression using bacterial expression vectors, and we report here the identification of a theoretically predicted hairpin structure in the mRNA corresponding to the N-terminal portion of the mature coding portion of bFSHbeta cDNA that is responsible for attenuating its expression in E. coli. When full-length FSHbeta was expressed using the bacterial expression vector, a very low expression was obtained. However, when fragments of FSHbeta with N-terminal deletions (amino acids 24-110 and 13-110) were expressed using the same expression strategy, a 30- to 40-fold higher expression was observed. This low expression of FSHbeta could be attributed to a hairpin structure present in the first 12 codons of mature FSHbeta mRNA. Disruption of this structure without changing the amino acid sequence resulted in a higher level of expression of FSHbeta. The predicted hairpin structure, though away from the transcriptional and translational start site, was able to downregulate the expression of FSHbeta probably by impeding the movement of ribosomes.