Use of synthetic ribosome binding site for overproduction of the 5B protein of Insertion sequence IS5

Abstract

Insertion sequence IS5 is a bacterial transposable element which contains three open reading frames designated 5A, 5B and 5C. Althoclh there was no detectable expression from the 5B open reading fra.ne when it was preceded by the native promoter and ribosome binding site or by a tac promoter and the native ribosome binding site, we have overproduced a 5B protein both in vitro and in Escherichia coli cells by using a tac promoter and a specially-designed synthetic ribosome binding site. 1-galactosidase fusion studies suggested that the synthetic binding site is at least 150-fold more efficient than the native binding site. The 5B protein amounted to 80-85% of the total protein made in vitro and 20-25% of the total protein pulse-labelled in whole cells. It is stable in vitro but rapidly degraded in vivo. Thus expression of the 5B gene appears to be limited by both poor translation initiation and protein degradation.

Transcript analysis ofEscherichia coliK-12 insertion element IS5

“…This observation is in accord with the findings of the current study and suggests that the ins5B gene probably does not have its own promoter. Moreover, recombinant overproduction of the 5B protein could only be achieved by creating an artificial ribosome‐binding site in front of the ins5B gene [9], which indicates that ins5C and ins5B are translationally coupled.…”

“…However, examination of the in vivo expression levels suggests that the ins5B gene is unlikely to be expressed from its own promoter [8]. Moreover, attempts to overproduce the 5B protein using the native ribosome‐binding site of the ins5B gene failed [9], suggesting that the ins5B gene is transcribed together with ins5C . Despite having been characterised more than 20 years ago, no detailed transcript analysis of IS 5 has been undertaken.…”

Mathematical modeling of translation initiation for the estimation of its efficiency to computationally design mRNA sequences with desired expression levels in prokaryotes

“…However, a few nucleotide changes around the start codon can dramatically affect translation efficiency and may alter protein expression levels by up to 250-fold [ 7 - 10 ]. Thus, if both transcription and translation processes are not considered during the design of synthetic networks, the realized networks could show unpredictable or unstable behavior [ 7 , 8 , 11 , 12 ]. In order to guarantee the robust operation of synthetic networks, the kinetics of both transcription and translation should be optimized, much in the way that nature has optimized biological systems through evolution [ 13 , 14 ].…”