Simple and Efficient Method for Heterologous Expression of Clostridial Proteins

Abstract

Many clostridial proteins are poorly produced in Escherichia coli. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli and the quantities of the corresponding tRNAs in E. coli are not sufficient to ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three E. coli genes, ileX, argU, and leuW, in E. coli; these genes encode tRNAs that are rarely used in E. coli (the tRNAs for the ATA, AGA, and CTA codons, respectively). Our data demonstrate that amplification of ileX dramatically increased the level of production of most of the clostridial proteins tested, while amplification of argU had a moderate effect and amplification of leuW had no effect. Thus, amplification of certain tRNA genes for rare codons in E. coli improves the expression of clostridial genes in E. coli, while amplification of other tRNAs for rare codons might not be needed for improved expression. We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence. Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E. coli.Clostridial proteins, such as tetanus toxin and seven serologically distinct botulinum neurotoxins (botulinum neurotoxin serotype A [BoNT/A], BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, and BoNT/G) that are produced by Clostridium tetani, Clostridium botulinum, Clostridium argentiensis, and Clostridium baratti, are powerful tools for studying the mechanisms of synaptic vesicle exocytosis (3,(20)(21)(22)(23). These toxins have been also used for therapeutic purposes, such as the treatment of strabismus, blepharospasms (24,25), and many other neurological conditions, as well as in clinical dermatology (4).Currently, BoNT/A and other clostridial neurotoxins and their fragments are purified from native Clostridium strains by using traditional purification protocols. Because these microorganisms are anaerobes, they pose technical problems. In addition, gene manipulation methods have not been developed for these microorganisms. Therefore, it has been difficult to construct Clostridium strains that produce derivatives of neurotoxins and other proteins. Genes for all eight clostridial neurotoxins have been cloned, and their sequences have been identified (2,6,9,18,30,31). Many attempts to express fragments of clostridial neurotoxins in Escherichia coli have failed because of the unusually high AT content of clostridial DNA. Makoff et al. successfully expressed a tetanus toxin fragment in E. coli (12) by optimizing sequences for codon usage in E. coli by complete synthesis of these sequences de novo. This approach, however, is very laborious and expensive.Recently, several groups of workers have demonstrated that rarely used codons can...

Genome of Bacteriophage P1

“…The ACA codon is the rarest threonine codon in E. coli, but its effect on translation has not been studied. The ATA codon, which is the fifth rarest codon in E. coli, is known to dramatically decrease translation of those E. coli mRNAs that contain it, especially when present in multiple copies, or in tandem, or in a single copy in the anterior part of a gene (99,166,353). The ATA codons are overrepresented more than twofold in 59 of 113 protein-coding genes of P1 as compared to their representation in E. coli genes.…”

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-β-1,3-glucanase bound to the outer cell surface

“…A succession of these codons in a heterologous gene impedes overexpression in E. coli host cells (Del Tito et al, 1995;Zdanovsky & Zdanovskaia, 2000). Accordingly, the heterologous expression of licA in E. coli was low and IPTG induction resulted in severe growth retardation and heavy protein degradation.…”

Improvement of Cellulolytic Properties of Clostridium cellulolyticum by Metabolic Engineering

“…No immunologically cross-reacting materials were detected in extracts from negative controls. Despite the presence of one rarely used codon in the pdc gene in C. cellulolyticum (Ͻ0.01%), i.e., the CGG arginine codon (frequencies of codon utilization are available at http:www.kazuka.or.jp/codon), PDC and ADH II were efficiently expressed in CC-pMG8 cells, as generally found with foreign genes cloned in Clostridium species (29) and in contrast to clostridial proteins poorly produced in E. coli (31). The results were confirmed by the quantification of Western blot signals, which revealed only 3-and 1.5-fold better production of PDC and ADH II in E. coli than in C. cellulolyticum.…”