Thermostable Artificial Enzyme Isolated by In Vitro Selection

Abstract

Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65°C. The most efficient enzyme, ligase 10C, was not only active at 65°C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

Genetic Code Evolution Investigated through the Synthesis and Characterisation of Proteins from Reduced‐Alphabet Libraries

“…These data support the idea that a protein does not have to display discrete secondary or compact tertiary structure to function. Furthermore, in earlier work, we generated an artificial RNA ligase enzyme that had a CD spectrum similar to all but one variant reported here and, yet, this protein had a stably folded three‐dimensional structure and a melting temperature of 72 °C. Importantly, the example of this unnatural enzyme highlights that, in contrast to common belief, the absence of canonical secondary structure elements, namely α‐helices or β‐strands, or the lack of a large compact hydrophobic core does not preclude a stable three‐dimensional structure and enzymatic activity.…”

“…Library variants were subjected to ANS binding, followed by excitation and fluorescence detection (Figure ). For comparison, we included two well‐folded proteins: ribonuclease A and BSA, as well as our artificial RNA ligase, which is a small 87‐residue protein that has an unusual three‐dimensional structure with high conformational dynamics . Of the 13 library variants examined, only variant 16‐1 exhibited fluorescence similar to that of the well‐folded control proteins (Figure ).…”

Genetically modified proteins: functional improvement and chimeragenesis

“…Beneficial mutations that lead to increased thermostability or decreased thermoactivity, improved pH-optimum or solubility can be involved in many different mechanisms determined by the laws of physics and chemistry including solvent interactions, structural support, and electrostatic balance. 59,94,95,96,97,98,99,100 Consequently, the search for variants with improved function is best treated as a combinatorial optimization problem, in which a number of parameters must be optimised simultaneously to achieve a successful outcome imitating the nature's manner.…”

Extensive libraries of gene truncation variants generated byin vitrotransposition

“…We removed the Flag tag at this step, as the original ligase 10C did not have this feature either (32) and the epitope tag was used here during mRNA display selection only for purification purposes. Thus, we obtained the ligase variants Del 13 ΔFlag, Del 18 ΔFlag and Del 18 I22T ΔFlag.…”