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Microbial Biotechnology (MB) | Protein Structure, Function, and Engineering
Microbiol. Biotechnol. Lett. 2022; 50(2): 240-244
Ho-Dong Lim, So-Young Han, Gi-Hye Park, Dae-Eun Cheong, and Geun-Joong Kim*
Department of Biological Sciences and Research Center of Ecomimetics, College of Natural Sciences, Chonnam National University, Gwangju 61186, Republic of Korea
Correspondence to :
Geun-Joong Kim, firstname.lastname@example.org
Precisely reliable and quantitative reporters can provide phenotypes that are consistent with research goals in protein expression. Here, we developed an improved reporter mATglu III 5 by directed evolution using a versatile β-glucosidase ATglu derived from Agrobacterium tumefaciens. When expressed in hosts, a vector containing this mutant distinctly showed a colored or fluorescent phenotype, according to the supplemented substrate, without any inducer. Analysis of mATglu III 5 showed it to be fully functional in fusion state with oligomeric proteins, especially under non-induction conditions, thereby offering an alternative to conventional reporters.
Keywords: Versatile reporter, β-glucosidase, directed evolution, activity staining
As an indispensable tool of biological research, reporter system can be used as a fundamental technique to detect or prove whether a gene of interest is expressed when it is introduced into the corresponding system. This is a crucial step if the expressed protein from the target gene could not be detected obviously by a typical phenotypic change such a difference in cell growth or morphology [1, 2]. Typically, successfully implemented reporters are mainly derived from green fluorescent protein GFP because it requires no auxiliary cofactors or substrates . However, it has the time-consuming maturation step and also needs an exposure to toxic UV for fluorescence emission. Thus, superfolder GFP  and mCherry  with improved functions have been developed and widely used.
As an alternative, β-galactosidase is a widely used reporter that can be easily detected by fluorogenic and chromogenic assay using specific substrates . However, an intrinsic drawback of
A β-glucosidase (ATglu) from
We here used directed evolution to obtain ATglu mutants (mATglu) with improved function as a reporter
To further analyze the expression patterns and relative activities of mutated enzymes, screened clones were inoculated into the same LB liquid medium and cultured at 37℃ and 200 rpm for 8 h. Resulting cells was reseeded (1%, v/v) and further grown in the same medium for 24 h under the conditions without any inducer. These cells were harvested by centrifugation at 10,000 g for 2 min and then washed with phosphate-buffered saline (PBS). After cell lysis by untrasonication, the supernatant was subjected to native-PAGE and zymogram analysis using three substrates (fluorescent MUG, 0.1 mM; chromogenic X-gal and indican, 1 mM) according to the previous study . As shown in Fig. 2A, mutated enzymes had higher activities on solid gel than the wild-type enzyme. Especially, zymogram activities of two mutants, III 4 and III 5, against X-gal and indican were the highest among clones. Active protein bands of the two mutants toward MUG were also higher than those of the wild type. We intentionally selected two clones (III 4 and III 5) for further analyses of expression level and solubility by SDS-PAGE using the same supernatant.
As shown in Fig. 2B, both mutants showed 2.8 to 3.3 folds increase in the expression level, resulting in proportional increases in soluble fractions. These differences in expression level and solubility were not observed under the induction condition with IPTG (data not shown). Additionally, only marginal increases (1.1- 1.3 folds) in specific activities of two mutant enzymes were detected by using indican and MUG as substrates. Therefore, activity differences in the screening step were mainly linked to expression level and solubility under non-induction conditions, not enhanced activity. This assumption is partly supported by the located mutation residing far away from the activity site (residues 355 to 365) of the related glycosyl hydrolase family I. These characters of the mutated enzymes were unexpected. However, they are suitable for monitoring protein expression as reporters under non-invasive (without any interference by a stressful inducer IPTG) conditions. Considering these points, we finally selected mutant III 5 as a plausible candidate for further analyses in terms of fusion ability with the target protein.
It is well-known that a reporter protein should have a suitable fusion ability to monitor the expression of the target protein
Two representative fluorescence reporters, GFPuv and DsRed, were also fused with III 5 to monitor whether III 5 could function in a fusion state with oligomeric proteins. The supernatant from cells expressing III 5- GFPuv fusion protein was prepared according to the same procedure above and loaded on a native-PAGE (8%), and further attempted to confirm activity toward 0.1 mM MUG. The resulting zymogram revealed clearly detectable active bands corresponding to the fusion protein on acrylamide gels. As a control, non-fused III 5 was also detected clearly in the corresponding size (Fig. 3B). We also examined fluorescence images of tetrameric DsRed-fused proteins in cells with a fluorescence microscope (Olympus, Japan) using cultivated cells . Expectedly, the same spots of two fluorescence, red from DsRed and blue from catalytic activity of III 5 toward MUG, overlapped (Fig. 3C), although a spot within a cell has more blue fluorescence and thus hardly detected red fluorescence due to slow maturation time  and lower molecular ratio of DsRed than MUG. These phenomena were further supported by fluorometric assay (Infinite M200, TECAN) using supernatant from the same culture. As shown in Fig. 3D, the emitting fluorescence from MUG was more proportionally increased than that of DsRed in the supernatant used.
Here we developed a reporter system using a directed evolved β-glucosidase, which is more reliable and less restrictive than the wild type enzyme. Although further validation in a practical study is needed, a versatile substrate spectrum and potential function of this reporter under non-induction condition could be used for various gene expression systems.
This work was supported by a grant (grant number: 20170305) from the Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries, Korea. This work was also supported by a grant (grant number: 2021R1A2C1006734) of the Basic Science Research program of the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology of Korea (MEST), Republic of Korea.
The authors have no financial conflicts of interest to declare.
Seung-Goo Lee , Eu-Gene Rha , Jae-Seok Ha , Jeong-Min Lee and Sun-Hwa KimMicrobiol. Biotechnol. Lett. 2008; 36(2): 149-157 https://doi.org/10.4014/mbl.2008.36.2.149
이대희, 김수경, 이승철, 윤병대, 황용일Microbiol. Biotechnol. Lett. 1995; 23(2): 145-149 https://doi.org/10.4014/mbl.19184.108.40.206