mRNA Display For Enzyme Engineering Service

mRNA Display is an ultra-high-throughput in vitro selection technique used for the directed evolution and engineering of enzyme variants. In this cell-free system, a covalent linkage is formed between an enzyme (phenotype) and its encoding messenger RNA (genotype) via a puromycin linker during in vitro translation. This creates a library of enzyme-mRNA complexes, where the genetic information of a given enzyme is physically attached to the enzyme itself. mRNA Display enables the screening of libraries with unparalleled diversity, often exceeding 10^12 variants, making it a powerful platform for discovering enzymes with rare or subtle functional improvements.

CD Biosynsis offers expert CRO services in mRNA Display for Enzyme Engineering, managing the entire process from massive library construction to functional selection and sequencing. Our in vitro system provides a level of control and speed unattainable by cell-based methods. We specialize in designing functional selection strategies, such as capture based on high-affinity transition state binding or activity-based covalent labeling, to isolate the best enzyme mutants. mRNA Display is particularly effective for generating enzymes with dramatically enhanced catalytic efficiency, modified substrate specificity, or improved stability for therapeutic and industrial applications.

Highlights

mRNA Display provides the highest possible throughput and speed for enzyme discovery and evolution.

• Ultra-High Diversity: Enables the screening of libraries up to 10^13 variants, ensuring near-complete coverage of sequence space for targeted regions.
• Phenotype-Genotype Link: Forms a robust covalent bond between the enzyme (phenotype) and its mRNA (genotype), guaranteeing efficient recovery and amplification.
• In Vitro Speed and Control: Selection cycles are fast and precisely controlled in a test tube, eliminating issues with cell toxicity, transformation efficiency, and expression variability.
• Selection for Activity: Highly amenable to activity-based selection methods, allowing for direct sorting based on catalytic turnover rather than just binding affinity.

Applications

mRNA Display is essential for high-impact enzyme engineering projects where vast diversity is required:

Maximum Catalytic Enhancement

Exploring vast sequence space to find rare mutants that significantly reduce the activation energy barrier for the reaction (high kcat/Km).

Engineering Promiscuity

Discovering enzymes capable of catalyzing entirely new, non-native reactions or accepting structurally diverse, non-natural substrates.

Therapeutic Enzyme Optimization

Generating stable, highly active enzyme variants for drug applications, such as enzyme replacement therapy or chemotherapy activation.

Stabilization and Solubility

Selecting variants that maintain function under harsh conditions (high temperature, organic solvents) or exhibit improved solubility and reduced aggregation.

Platform

Our mRNA Display platform leverages cutting-edge molecular biology for selection accuracy and speed.

Cell-Free In Vitro Translation

Uses highly efficient rabbit reticulocyte or wheat germ cell-free systems, maximizing the yield of functional protein-mRNA complexes.

Activity-Based Selection

Design of selection steps that require the enzyme to perform a catalytic step, often involving covalent labeling with an activated substrate or transition state analog.

High-Stringency Panning

Sequential rounds of selection with increasing stringency (e.g., lower target concentration, faster off-rates) to enrich for only the highest-performing mutants.

Rapid PCR Amplification

Efficient conversion of the selected mRNA back into cDNA (genotype) via reverse transcription-PCR for amplification and subsequent rounds of selection.

Next-Generation Sequencing (NGS)

Deep sequencing of enriched pools to identify the consensus sequences and quantify the enrichment ratio of successful variants.

Workflow

Our mRNA Display workflow is an efficient cycle performed entirely in vitro to maximize throughput:

• Library Preparation: Create the DNA library (e.g., using error-prone PCR or saturation mutagenesis) and transcribe it in vitro to generate the mRNA library.
• In Vitro Translation and Complex Formation: Translate the mRNA library in a cell-free system containing a puromycin linker, which covalently fuses the newly synthesized enzyme to its encoding mRNA.
• Selection (Panning): Incubate the enzyme-mRNA complexes with the immobilized target (e.g., substrate analog, inhibitor, or capture agent). Use stringent washing to remove non-binders.
• Elution and Recovery: Elute the selected complexes, typically by heating or chemical cleavage. Recover the mRNA by reverse transcription (RT).
• Amplification and Iteration: Amplify the recovered cDNA using PCR and use it as the template for the next round of in vitro transcription/translation (typically 4-6 rounds).
• Final Analysis: Sequence the final enriched pool using NGS and functionally validate individual clones by expressing and purifying the encoded enzyme.

CD Biosynsis delivers the highest-performance enzyme variants discovered from ultra-large libraries. Every project includes:

• Detailed Selection Report: Documentation of library size, selection stringency, and enrichment metrics across all rounds.
• Enriched Variant Sequences: DNA and protein sequences of the top enzyme hits identified by NGS and confirmed by Sanger sequencing.
• Functional Kinetics: Purified protein kinetic data (Km, kcat, selectivity ratio) comparing the top mutant to the wild-type enzyme.
• Cloning Vector: Delivery of the plasmid containing the optimized enzyme sequence ready for large-scale expression.