Anti-Coagulant and Antimicrobial Recombinant Heparin-Binding Major Ampullate Spidroin 2 (MaSp2) Silk Protein.

Pranothi Mulinti,Kristen Kurtzeborn,David W Grainger,Amanda E Brooks,Narayanaganesh Balasubramanian,Shane J Stafslien,Dorina Diekjürgen

doi:10.3390/bioengineering9020046

Pranothi Mulinti, Kristen Kurtzeborn + Show 5 more

Open Access

https://doi.org/10.3390/bioengineering9020046

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Abstract

Governed by established structure–property relationships, peptide motifs comprising major ampullate spider silk confer a balance of strength and extensibility. Other biologically inspired small peptide motifs correlated to specific functionalities can be combined within these units to create designer silk materials with new hybrid properties. In this study, a small basic peptide, (ARKKAAKA) known to both bind heparin and mimic an antimicrobial peptide, was genetically linked to a protease-resistant, mechanically robust silk-like peptide, MaSp2. Purified fusion proteins (four silk domains and four heparin-binding peptide repeats) were expressed in E. coli. Successful fusion of a MaSp2 spider silk peptide with the heparin-binding motif was shown using a variety of analytical assays. The ability of the fusion peptide to bind heparin was assessed with ELISA and was further tested for its anticoagulant property using aPTT assay. Its intrinsic property to inhibit bacterial growth was evaluated using zone of inhibition and crystal violet (CV) assays. Using this strategy, we were able to link the two types of genetic motifs to create a designer silk-like protein with improved hemocompatibility and antimicrobial properties.

Highlights

A hierarchical structure rooted in the biochemical composition of two main proteins, MaSp1 and major ampullate spidroin 2 (MaSp2), gives major ampullate spider silk an impressive balance of strength and elasticity, unrivaled by synthetic materials [1,2]
In addition to mechanical properties that can be manipulated by genetically combining functional motifs, several recombinant spider silk fusion proteins have been explored to date
A new protein fusion comprising spider silk-derived MaSp2 peptides genetically linked with a consensus heparin-binding peptide was shown to bind heparin both at a surface and in buffer solution

Summary

Introduction

A hierarchical structure rooted in the biochemical composition of two main proteins, MaSp1 and MaSp2, gives major ampullate spider silk an impressive balance of strength and elasticity, unrivaled by synthetic materials [1,2]. Phylogenetic analyses of major ampullate silk sequences, in addition to other proteomic [3] and structural analyses [4,5], revealed evolutionarily conserved repetitive amino acid motifs subsequently correlated with specific structural and functional features of the silk [6]. These correlative conclusions have been well-studied by producing clones of simplified monomer motifs [4,7,8,9] to identify and establish overarching structure–property relationships [6,10]. This manuscript describes a new genetic chemically complexed with heparin and chitosan to create an anticoagulant, antimicrobial, modification of silk designed to both capture circulating heparin and provide intrinsic dual-function protein [17]

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