Chemie  |  Biochemie  |  Medizin

 

Leandro Fondado, 2006 | Aarau, AG
Benjamin Meyer, 2006 | Schöftland, AG

 

Wound adhesives are an important surgical tool to quickly close wounds through polymerization. However, they often cause adverse reactions, have suboptimal mechanical properties, and fail in wet environments. To reduce immunogenicity and more closely mimic natural healing, we propose the human calcium-binding protein S100G as a novel polymerization module. We designed fusion proteins incorporating multiple S100G domains and an extracellular matrix (ECM)-binding component to enable calcium-dependent polymerization and adhesion to wound surfaces. Thirteen fusion proteins were successfully produced in Escherichia coli. However, polymerization assays showed no signs of large-scale polymerization. Thus, a functional polymerization system based on S100G could not be established. The observed oligomerization suggests undesired domain interactions, potentially due to suboptimal protein design, warranting further research.

Introduction

Current wound adhesives, both synthetic and biological, carry risks of foreign body reactions and infections. An adhesive based on human proteins could greatly reduce these risks.
S100G is a human, calcium-binding protein involved in the intestinal absorption of calcium ions. After cleavage into its two domains (CN and CC), it reassembles in calcium-rich environments. This property was previously used to restore function to a split green fluorescent protein.
We hypothesize that by concatenating multiple CN or CC domains in a single protein, these domains could be used as a calcium-dependent cross-linking agent, resulting in polymerization. Fusion with ECM-binding proteins could allow the polymer to adhere to wound surfaces.
To verify this hypothesis, we aimed to answer the following questions:

1. Were our fusion proteins produced correctly?
2. Can S100G be used as a polymerization module in our fusion proteins?
3. How does the interdomain linker affect polymerization?
4. Do our constructs adhere to ECM?

Methods

Thirteen fusion proteins were recombinantly produced in E. coli. This included flexibly and rigidly linked chains of 4-16 CN or CC domains, as well as ECM-binding proteins attached to two CN domains. SDS-PAGE and DNA sequencing were used to determine correct production of proteins.
Polymerization was assessed by a visual assay and dynamic light scattering, which measures particle size. A GFP-based assay was developed to test domain interactions and adhesion to the ECM.

Results

Most proteins were produced successfully according to SDS-PAGE at 2–5 mg/mL with an estimated purity of 50–90% based on SDS-PAGE. One rigid CC construct showed low yield and purity, likely due to protein instability. The rigidly linked proteins had lower solubility, leading to a tendency to precipitate. Protein folding was not verified.
No macroscopic changes were observed in the visual assay. The GFP assay showed no detectable staining of the cells, though no positive controls were used.
Dynamic light scattering showed an approximately tenfold increase in particle size upon calcium addition. This indicates oligomerization, as true polymerization would have resulted in a greater size increase. Unexpectedly, this increase was observed in samples containing both complementary and identical S100G domains.

Discussion

The results indicate that our fusion proteins are unsuitable for polymerization in their current state. While misfolding cannot be excluded, the observed oligomerization suggests that some interactions are taking place. These interactions may be too weak to sustain a polymer network, either due to the properties of the S100G domain assembly or to the design of the fusion proteins. No clear differences between the tested linker systems were found, except for the reduced solubility of rigidly linked proteins.
The dynamic light scattering results point to some unexpected intramolecular interactions between identical domains within the same fusion protein.
The GFP-based assay requires refinement to assess adhesion of the proteins to the ECM, such as through the inclusion of positive controls.

Conclusions

Although polymerization was not achieved, S100G-based constructs showed calcium-dependent oligomerization. Further optimization of the assays, construct design, and addition of a protein folding assay are required. Another possible approach is mutagenesis of the individual S100G domains to increase their binding strength or otherwise increase their viability as polymerization modules. If successful, our strategy may ultimately provide a novel tool for closing wounds.

 

 

Würdigung durch den Experten

Prof. Dr. Peter Kast

Das Projekt von Leandro Fondado und Benjamin Meyer verfolgte experimentelle Ansätze zur Entwicklung von Wundklebstoffen basierend auf fusionierten Proteindomänen. Sie entwarfen, produzierten und charakterisierten dabei neuartige Fusionsproteine mittels moderner molekularbiologischer Strategien. Obwohl die sehr ambitionierten Designs nicht wie geplant funktionierten, beeindruckt diese Arbeit mit der Herangehensweise, der Stringenz der Dateninterpretation, den Verbesserungsvorschlägen und dem bemerkenswerten Einsatz und der wissenschaftlichen Reife bei der Umsetzung der verlangten Anpassungen.

Prädikat:

Gold

Sonderpreis «Mostratec» gestiftet von der SJf-Trägerschaft

 

 

 

Alte Kantonsschule Aarau
Lehrer: Dr. Michael Kappeler