High-yield production of a super-soluble miniature spidroin for biomimetic high-performance materials

Benjamin Schmuck,Gabriele Greco,Andreas Barth,Nicola M Pugno,Jan Johansson,Anna Rising

doi:10.1016/j.mattod.2021.07.020

Abstract

The mechanical properties of artificial spider silks are approaching a stage where commercial applications become realistic. However, the yields of recombinant silk proteins that can be used to produce fibers with good mechanical properties are typically very low and many purification and spinning protocols still require the use of urea, hexafluoroisopropanol, and/or methanol. Thus, improved production and spinning methods with a minimal environmental impact are needed. We have previously developed a miniature spider silk protein that is characterized by high solubility in aqueous buffers and spinnability in biomimetic set-ups. In this study, we developed a production protocol that resulted in an expression level of >20 g target protein per liter in an Escherichia coli fed-batch culture, and subsequent purification under native conditions yielded 14.5 g/l. This corresponds to a nearly six-fold increase in expression levels, and a 10-fold increase in yield after purification compared to reports for recombinant spider silk proteins. Biomimetic spinning using only aqueous buffers resulted in fibers with a toughness modulus of 74 MJ/m3, which is the highest reported for biomimetically as-spun artificial silk fibers. Thus, the process described herein represents a milestone for the economic production of biomimetic silk fibers for industrial applications.

Highlights

The favorable mechanical properties and the biocompatible and biodegradable nature [1,2,3,4] of spider silk have triggered a quest to harness this material for various industrial applications
The culture was grown at 25 °C, before the temperature was decreased to 20 °C when optical density at 600 nm (OD600) reached 50, and protein expression was induced with IPTG (150 mM)
Compared to native dragline silk, the toughness modulus of NT2RepCT fibers is around 50%, and compared to artificial silk fibers that have been produced from recombinant spidroins with reported expression levels >1.5 g/l, the toughness modulus is equal or significantly higher (Fig. 1e)

Summary

Introduction

The favorable mechanical properties (high strength, yet extensible) and the biocompatible and biodegradable nature [1,2,3,4] of spider silk have triggered a quest to harness this material for various industrial applications. In addition to the relatively low yields, the recombinant spidroins are prone to aggregate, which is why common techniques to prepare the spinning solutions (dopes) include the use of denaturing agents like urea/ guanidium for the re-suspension of inclusion bodies, hexafluoroisopropanol (HFIP) for solubilizing lyophilized proteins, and methanol/isopropanol as a coagulation agent for spinning [14,19,22,23] Fibers produced using these methods have reached the toughness modulus of native spider silk, provided that additional manual post-spin stretching is applied [14,19,22]. Bacterial shake-flask cultivations employing the standard E. coli BL21 (DE3) strain express NT2RepCT with a yield in a range above 100 mg/l [26], which is a good starting point for further optimizations

Results and discussion

Concluding remarks

Materials and methods

Funding details