Cell Surface Display For Enzyme Engineering Service

Cell Surface Display (CSD) is a versatile technology for enzyme engineering that anchors enzyme variants onto the outer membrane of a host cell (such as E. coli, yeast, or mammalian cells). This technology genetically links the enzyme (phenotype) to its encoding DNA inside the host cell (genotype), allowing large libraries to be screened using fluorescence-activated cell sorting (FACS). CSD is particularly advantageous for engineering enzymes designed to function in a cellular environment, facilitating selection for improved activity, stability, and substrate specificity in a relevant biological context.

CD Biosynsis offers comprehensive CRO services in Cell Surface Display for Enzyme Engineering, utilizing diverse host platforms tailored to the enzyme's complexity and end application. We employ various anchoring motifs (e.g., Lpp-OmpA, Ice Nucleation Protein, Aga2p) to achieve high-density, functional display. Our services are ideal for evolving complex enzymes, including those requiring disulfide bonds or cofactors, and for screening vast mutant libraries (up to 10^8 cells). By integrating robust library generation with high-resolution FACS screening using fluorescent substrates, we efficiently identify and recover superior enzyme mutants with highly tailored properties.

Highlights

Cell Surface Display offers a versatile and high-throughput system for enzyme evolution in a cellular context.

• Host Versatility: Can be implemented in prokaryotic (E. coli) or eukaryotic (Yeast, Mammalian) systems, allowing optimization in a relevant biological environment.
• FACS Compatibility: Enables quantitative, high-resolution screening and sorting of millions of individual cells based on enzyme activity or binding affinity.
• Phenotype-Genotype Link: The enzyme displayed on the surface is directly linked to the plasmid DNA within the cell, facilitating easy recovery of the genetic sequence.
• Complex Protein Folding: Eukaryotic hosts (Yeast/Mammalian) support proper disulfide bond formation and post-translational modifications, suitable for complex enzymes.

Applications

CSD is a critical tool for developing complex and environmentally functional enzymes:

Whole-Cell Biocatalysis

Developing engineered cells for direct use in industrial processes, where the displayed enzyme acts on substrates in the medium.

Environmental Biosensors

Engineering cells to display enzymes that detect and signal the presence of specific environmental toxins or chemicals.

Targeted Drug Delivery

Displaying enzymes on the surface of therapeutic cells to modulate the local environment or degrade toxic compounds in vivo.

Affinity and Stability Optimization

Precisely tuning the enzyme's binding affinity (Km) or selecting variants with superior thermal or pH stability in a high-throughput format.

Platform

Our CSD platform leverages high-resolution sorting and robust anchor systems across various host species.

Custom Host Selection

Selection between E. coli CSD (high speed), Yeast Surface Display (eukaryotic folding), or Mammalian CSD (therapeutic relevance) based on project needs.

High-Density Anchoring

Utilization of optimized anchor proteins (e.g., E. coli OmpA, yeast Aga2p) to achieve maximum display density and reliable quantification.

Multi-Color FACS Screening

Simultaneous measurement of cell viability, display level, and enzyme activity using multiple fluorescent probes for high-precision sorting.

Kinetic Selection Protocols

Implementing selection protocols (e.g., varying substrate concentration, time-lapse incubation) to directly screen for improved kcat or Km rather than total product yield.

Whole-Cell Activity Assays

Validation of the best mutants by performing enzymatic assays directly on the whole-cell biocatalyst before purification.

Workflow

Our Cell Surface Display engineering service follows an iterative cycle for efficient library screening and selection:

• Vector Design and Library Generation: Clone the enzyme mutant library into the CSD vector, ensuring fusion with the selected anchor protein and a reporter tag.
• Cell Transformation and Display: Transform the chosen host cells (e.g., E. coli, Yeast) and induce the expression of the enzyme on the cell surface.
• Fluorescent Labeling and Staining: Label the cell population with a probe for the reporter tag (total display) and a fluorescent substrate or activity probe (function).
• FACS Sorting: Sort the cells using FACS, selecting for the highest ratio of activity signal to display signal (high specific activity).
• Mutant Recovery and Amplification: Recover the sorted cells, extract the plasmid DNA, and amplify the gene encoding the successful variants for the next round.
• Iterative Rounds and Final Analysis: Repeat the sorting (typically 3-4 rounds) to maximize enrichment. Sequence the resulting clones and characterize the purified enzyme.

CD Biosynsis delivers functionally superior enzyme variants optimized for cellular applications. Every project includes:

• CSD Design Report: Documentation of the anchor system, host details, and FACS gating strategy used for selection.
• Engineered Clone: Delivery of the plasmid containing the optimized enzyme sequence, verified by sequencing.
• Functional Characterization: Quantitative data (Km, kcat) comparing the performance of the engineered enzyme versus the wild-type.
• Validated Cells: Upon request, delivery of the final engineered host cells ready for whole-cell biocatalysis testing.