Thermostable lichenase as a translational reporter

Abstract

Hybrid genes containing the reporter gene for thermostable lichenase and model genes rec A, rec A1, cry 3a, cry 3aM, and ssp 1 were constructed. The expression of these genes was studied in prokaryotic and eukaryotic cells. The presence of lichenase in the hybrid proteins was shown to facilitate analysis of the hybrid protein expression in transgenic organisms. Owing to high relative activity and thermostability of lichenase, the activity of this enzyme can be measured by simple, rapid and sensitive qualitative and quantitative methods that do not require costly equipment and reagents. Using the zymograms method, molecular masses of the lichenase-containing hybrid proteins can be precisely estimated. This method is proposed instead of Western blotting using lichenase as a translational reporter. Our results showed that the use of thermostable lichenase as a translational reporter yields the data that are problematic to obtain using traditional methods of gene expression analysis, which is of importance for fundamental and applied research. MOLECULAR GENETICSRUSSIAN JOURNAL OF GENETICS Vol. 41 No. 1 2005 KOMAKHIN et al .

Modification of the Sunflower Defensin SD2 Gene Sequence and Its Expression in Bacterial and Yeast Cells

“…The use of thermostable lichenase as a convenient translation reporter was based on our data on the construction and expression of the lichenase-based hybrid proteins [15]. It was demonstrated that the presence of lichenase in the hybrid proteins facilitated analysis of the hybrid proteins expression in transgenic organisms.…”

“…It was demonstrated that the presence of lichenase in the hybrid proteins facilitated analysis of the hybrid proteins expression in transgenic organisms. Furthermore, in the hybrid proteins lichenase, as well as the proteins of interest, preserved their properties [15].…”

From the mechanisms of genetic transposition to the functional genomics

“…In addition, the constructed vector system helps to obtain data on regulatory functions of noncoding sequences by simple and fast qualitative and quantitative methods, which are inexpensive and less time-consuming (Table 2). The thermostable lichenase served both as a transcription reporter used to study the regulation of gene expression at the level of transcription and as a suitable translation reporter in studies on the expression of certain model genes [82]. The results obtained indicated that the use of thermostable lichenase as a translation reporter substantially facilitates analysis of hybrid protein expression and assists obtaining the data, which problematic or impossible to obtain by traditional methods in analysis of gene expression.…”

“…The results obtained indicated that the use of thermostable lichenase as a translation reporter substantially facilitates analysis of hybrid protein expression and assists obtaining the data, which problematic or impossible to obtain by traditional methods in analysis of gene expression. Additionally, the presence of lichenase in hybrid proteins, e.g., δ -endotoxin and recombinase, allows transgene monitoring in agrocenosis by fast and sensitive methods of analysis of lichenase expression [82].…”