Tetsuya Kamioka

Experimental Identification and Theoretical Analysis of a Thermally Stabilized Green Fluorescent Protein Variant

In this study, we aim to relate experimentally measured macroscopic properties to dynamic and structural changes as calculated by molecular dynamics (MD) simulations. We performed the analysis on four GFP (green fluorescent protein) variants, which have amino acid replacements or insertion in a flexible region on the protein surface and which resulted from a previous protein splicing reaction optimization experiment. The variants are a reference GFP (CEGFP), GFP-N144C, GFP-N144C/Y145F, and a GFP with five residues inserted between Y145 and N146 (GFP-5ins). As a result, we identified a single Y145F mutation that increased the thermal stability of GFP-N144C/Y145F by 3-4 °C. Because circular dichroism measurements indicated that the overall GFP β-barrel fold was maintained in all variants, we presumed that the fluorescence activity and thermal stability related to local changes that could be detected by standard MD simulations. The 60 ns MD simulations indicated that the Y145s hydroxyl group, which is straight and buried in the crystal structure, was bent avoiding the hydrophobic core during the simulation in both CEGFP and GFP-N144C. This local strain was relieved in GFP-N144C/Y145F, where the tyrosines hydroxyl group was replaced with the F145 hydrophobic aliphatic carbon. F145 remained indeed buried during the simulation maintaining local compactness, which presumably reflected the improved thermal stability of GFP-N144C/Y145F. Furthermore, the analysis of internal water molecules localized within the GFPs β-barrel suggested that a change in the local hydrogen bonding pattern around the chromophore correlated with a strong fluorescence activity decrease in GFP-5ins. Although relating experimental observation with calculated molecular features proved to be delicate, this study suggested that some microscopic features could be useful reporters for redesigning GFPs and other proteins. The newly identified GFP-N144C/Y145F was among the most stable GFP variant and demonstrates the potential of such computer-aided design.

Extraction of recombinant protein from Escherichia coli by using a novel cell autolysis activity of VanX.

Escherichia coli is a versatile, low-cost, and popular host for expressing recombinant proteins. However, extracting recombinant proteins from E. coli requires cell wall breakage, which is both time- and effort-consuming. Here we report a novel cell breakage method based on our recent finding that VanX, which is a d-Ala-d-Ala dipeptidase encoded in a vancomycin-resistant VanA gene cluster, exhibits a strong cell lysis activity when expressed in isolation in E. coli. In our strategy, we coexpress VanX with the target protein, causing cell autolysis and release of the cellular content into the culture medium. We demonstrated this strategy for two model proteins, a green fluorescent protein variant (GFPuv) and Gaussia luciferase, and optimized the autolysis conditions and coexpression vectors. The fluorescence activity of GFPuv collected from the medium was identical to that of GFPuv purified by conventional methods. Cell breakage by VanX-mediated autolysis is very simple to implement and will efficiently complement traditional methods.

Improved protein splicing reaction for low solubility protein fragments without insertion of native extein residues

Application of trans protein splicing has been limited both by solubility problems and by the insertion of native extein residues (NERs) at the splicing site. Here, we report two simple methods for overcoming these problems and increasing the yield and activity of the spliced product. First, low solubility was alleviated by adding arginine to the reaction buffer and optimizing the splicing reaction condition. The protocol was demonstrated in the context of a Green Fluorescent Protein variant (GFPuv), and the final yield was increased by 1.9‐fold compared to control experiments performed under the same conditions but without addition of arginine. Second, the insertion of NERs was overcome by mutating, instead of inserting, a minimal number of residues in the target protein to amino acids required for the splicing reaction. We identified optimal splicing sites that conserve as much as possible the prerequisite NERs. As a result, the GFPuv residues 142–146 (EYNYN) were mutated to the reportedly minimal required NERs, EYCFN. GFPs spliced using this strategy had no NERs insertion and a fluorescence activity six times stronger than a control GFPuv with five NERs inserted at the splicing site (residue 145/6). In principle, the present protocol (Sw/oNI) can be applied to any target protein, even when no sequence similarity to NERs is present, though it will introduce up to five mutations at the splicing site.

Biochemical and Biophysical Characterization of an Unexpected Bacteriolytic Activity of VanX, a Member of the Vancomycin-resistance vanA Gene Cluster

Background: VanX belongs to the vanA gene cluster, which confers vancomycin resistance by protecting the bacterial cell wall. Results: Bacteriolysis occurred when isolated VanX was expressed in E. coli, which originated from the dipeptidase activity of VanX. Conclusion: This is the first direct characterization of VanX-mediated bacteriolysis. Significance: This finding suggests a new class of therapeutic agents taking advantage of VanX-mediated bacteriolysis. VanX is a d-alanyl-d-alanine (d-Ala–d-Ala) dipeptidase encoded in the vancomycin-resistance vanA gene cluster. Here we report that strong bacteriolysis occurred when isolated VanX was expressed in Escherichia coli at temperatures lower than 30 °C, which was unexpected because the vanA operon confers vancomycin resistance by protecting the cell wall. Therefore, we monitored cell lysis by measuring sample turbidity with absorbance at 590 nm and VanX expression using SDS-PAGE. No cell lysis was observed when VanX was expressed, even in large quantities, in the cell inclusion bodies at 37 °C, suggesting that a natively folded VanX is required for lysis. In addition, VanX mutants with suppressed dipeptidase activity did not lyse E. coli cells, confirming that bacteriolysis originated from the dipeptidase activity of VanX. We also observed shape changes in E. coli cells undergoing VanX-mediated lysis with optical microscopy and classified these changes into three classes: bursting, deformation, and leaking fluid. Optical microscopic image analysis fully corroborated our interpretation of the turbidity changes in the samples. From a practical perspective, the finding that VanX expressed in isolation induces cell lysis suggests that inhibitors of VanA and VanH that act downstream from VanX could provide a new class of therapeutic chemicals against bacteria expressing the vancomycin-resistance gene cluster.

A novel screening system based on VanX mediated autolysis ‐ application to Gaussia luciferase

We report a novel bacterial screening protocol based on co‐expressing the target protein with VanX, an enzyme which mediates Escherichia colis autolysis and the release of the target protein into the culture medium, thereby facilitating activity measurement and screening from crude medium. This protocol as assessed with 19 Gaussia luciferase (GLuc) expressing colonies, was able to detect bioluminescence wavelength shift as small as 1.5 nm. We demonstrate the performance and versatility of this protocol by applying it to a semi‐rational search for GLuc variants with red‐shifted bioluminescence. Six GLucs sites, F113, I114, W143, L144, A149, and F151, were randomly mutated, and for each site, 50 colonies were cultivated in 3 mL samples, from which bioluminescence was measured without purification. We identified two GLuc single mutation red‐shifted variants: W143V and L144A. Their red shifted bioluminescence and biophysical/biochemical properties were confirmed using HPLC purified variants. Biotechnol. Bioeng. 2016;113: 1413–1420.