Molecular Determinants for Protein Stabilization by Insertional Fusion to a Thermophilic Host Protein

Tullman

Nicholes

et al. 2016

Biotechnol Biofuels

BackgroundSaccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose.ResultsThe pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA–XBP insertion library, which was used to transform E. coli strain JW3538-1 lacking the XBP gene. Screening for active XBP was based on the expression of GFP from the pT7T3GFP_XBP plasmid under the control of a xylose inducible promoter. In the presence of xylose, cells harboring a functional XBP domain in the fusion protein (XBP+) showed increased GFP fluorescence and were selected using FACS. The XBP+ cells were further screened for xylanase activity by halo formation around xylanase producing colonies (XynA+) on LB-agar-xylan media after staining with Congo red. The xylanase activity ratio with xylose/without xylose in supernatants from the XBP+/XynA+ clones was measured against remazol brilliant blue xylan. A clone showing an activity ratio higher than 1.3 was selected where the XynA was inserted after the asparagine 271 in the XBP, and this chimera was denominated as XynA–XBP271. The XynA–XBP271 was more stable than XynA at 55 °C, and in the presence of xylose the catalytic efficiency was ~3-fold greater than the parental xylanase. Molecular dynamics simulations predicted the formation of an extended protein–protein interface with coupled movements between the XynA and XBP domains. In the XynA–XBP271 with xylose bound to the XBP domain, the mobility of a β-loop in the XynA domain results in an increased access to the active site, and may explain the observed allosteric activation.ConclusionsThe approach presented here provides an important advance for the engineering enzymes that are stimulated by the final product.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0529-7) contains supplementary material, which is available to authorized users.

Section: Resultsmentioning

confidence: 99%

A xylose-stimulated xylanase–xylose binding protein chimera created by random nonhomologous recombination

Tullman

Nicholes

et al. 2016

Biotechnol Biofuels

“…More recently, a series of studies performed by Kim and coworkers demonstrated that domain insertion of a guest protein may benefit from high thermostability of a host protein [38,39,40,41]. Insertional fusion has recently been highlighted as a novel means of creating multi-domain protein complexes, where functionalities are often integrated and coupled with each other.…”

Section: Thermophilic Proteins As a Scaffold For Functional Evolutionmentioning

confidence: 99%

“…To overcome such energetic penalty, Kim and coworkers used a thermophilic maltodextrin-binding protein from Pyrococcus furiosus as a host protein, into which various guest enzyme domains, such as exoinulinase, TEM-1 β-lactamase and xylanase, were successfully inserted. The insertional fusions led to the creation of chimeric protein complexes, where thermostability of guest enzyme domains was improved by various mechanisms [38,39,40,41,42]. In contrast, similar insertional fusion into a mesophilic maltodextrin-binding protein from E. coli lowered thermostability and expression levels of a guest enzyme domain [39].…”

Section: Thermophilic Proteins As a Scaffold For Functional Evolutionmentioning

confidence: 99%

Thermophilic Proteins as Versatile Scaffolds for Protein Engineering

Finch

Kim

2018

Microorganisms

Literature from the past two decades has outlined the existence of a trade-off between protein stability and function. This trade-off creates a unique challenge for protein engineers who seek to introduce new functionality to proteins. These engineers must carefully balance the mutation-mediated creation and/or optimization of function with the destabilizing effect of those mutations. Subsequent research has shown that protein stability is positively correlated with “evolvability” or the ability to support mutations which bestow new functionality on the protein. Since the ultimate goal of protein engineering is to create and/or optimize a protein’s function, highly stable proteins are preferred as potential scaffolds for protein engineering. This review focuses on the application potential for thermophilic proteins as scaffolds for protein engineering. The relatively high inherent thermostability of these proteins grants them a great deal of mutational robustness, making them promising scaffolds for various protein engineering applications. Comparative studies on the evolvability of thermophilic and mesophilic proteins have strongly supported the argument that thermophilic proteins are more evolvable than mesophilic proteins. These findings indicate that thermophilic proteins may represent the scaffold of choice for protein engineering in the future.

“…Furthermore, different from end-to-end fusion in which domains are linked by a single contact point, when two domains are fused by domain insertion, they are linked by peptidyl bonds at two contact points with more intimate connection between the two proteins. This double contact can increase the protein stability of generated chimeras [23, 35, 40, 41], and the intimate connection can be used to couple the functions of two fused proteins.…”

Section: Engineering Switches By Protein Domain Insertionmentioning

confidence: 99%

Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion

Amarelle

BioMed Research International

et al. 2019

All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an “off” to an “on” state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.