Enhanced expression of tandem multimers of the antimicrobial peptide buforin II in Escherichia coli by the DEAD-box protein and trxB mutant.
The tandem multimeric expression of various peptides has been explored by many researchers. However, expression levels have usually not been proportional to the degree of multimerization. To increase the expression level in Escherichia coli of tandem multimers of a cationic antimicrobial peptide, buforin II, fused to an anionic peptide, we studied the effect of the DEAD-box protein and the trxB mutant on the expression of tandem multimers. An expression vector with a tac promoter was more effective in directing multimeric expression than one with a T7 promoter. The expression level of large multimers was substantially increased with the tac promoter, possibly through stabilization of long transcripts by synchronization of transcription and translation. Coexpression of the DEAD-box protein, an RNA-binding protein, with the T7 expression system increased the expression level of multimers, especially large multimers, due to protection of the long RNA transcripts. In addition, the use of the trxB mutant also enhanced the expression level of tandem multimers, which contain two cysteine residues at both ends of the monomeric unit. It seems that disulfide bonds formed in the multimers in the trxB mutant might help efficient charge neutralization for inclusion body formation of the multimers, resulting in enhancement of expression. Our results show that the expression of multimers can be improved through the stabilization of the long transcripts by the DEAD-box protein or the expression, under an oxidizing environment, of the trxB mutant in which covalent cross-links through disulfide bonds facilitate inclusion body formation of the multimeric fusion peptide.
Lee
J H
JH
Samyang Genex Biotech Research Institute, 63-2 Hwaam-dong, Yusung-gu, Taejon 305-348, Korea.
Kim
M S
MS
Cho
J H
JH
Kim
S C
SC
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D009154:Mutation; D010455:Peptides; D011506:Proteins / Q000235:genetics*; D020365:RNA Helicases / Q000235:genetics*; D020080:Tandem Repeat Sequences
D000900:Anti-Bacterial Agents; D010455:Peptides; D011506:Proteins; C097849:buforin II; D020365:RNA Helicases