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 coli's 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.

Biochemical and biophysical characterization of an unexpected bacteriolytic activity of VanX, a member of the vancomycin-resistance vanA gene cluster.

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.

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.

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).

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.