Transglutamination allows production and characterization of native‐sized ELPylated spider silk proteins from transgenic plants

Abstract

SummaryIn the last two decades it was shown that plants have a great potential for production of specific heterologous proteins. But high cost and inefficient downstream processing are a main technical bottleneck for the broader use of plant-based production technology especially for protein-based products, for technical use as fibres or biodegradable plastics and also for medical applications. High-performance fibres from recombinant spider silks are, therefore, a prominent example. Spiders developed rather different silk materials that are based on proteins. These spider silks show excellent properties in terms of elasticity and toughness. Natural spider silk proteins have a very high molecular weight, and it is precisely this property which is thought to give them their strength. Transgenic plants were generated to produce ELPylated recombinant spider silk derivatives. These fusion proteins were purified by Inverse Transition Cycling (ITC) and enzymatically multimerized with transglutaminase in vitro. Layers produced by casting monomers and multimers were characterized using atomic force microscopy (AFM) and AFM-based nanoindentation. The layered multimers formed by mixing lysine-and glutamine-tagged monomers were associated with the highest elastic penetration modulus.

Low-Tech, Pilot Scale Purification of a Recombinant Spider Silk Protein Analog from Tobacco Leaves

“…[16] for details). Briefly, a Major Ampullate Spidroin 1 derived gene (MaSp1), originated from the appropriate Nephila clavipes sequence, was designed to allow in vitro post-translational multimerization by a microbacterial transglutaminase.…”

“…Nicotiana tabacum is generally recognized as safe (GRAS) with no risk of cross-contaminating the food chain (Available online: ; accessed on 18 August 2016). Nicotiana tabacum has already been used for the production of antibodies, vaccines and spider silk analogs [13,14,15,16]. Plant-based expression systems are able to provide complex, correctly folded and post-translationally modified proteins [17].…”

“…The purification method based on this behavior is called Inverse Transition Cycling (ITC) [21]. After induced agglomeration, target proteins can be separated either via centrifugation (cITC) or a membrane-based (mITC) procedure [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. The spider silk analogs that are the focus of the present investigation contain such an ELP tag (see Figure 1A) and were previously purified via mITC [16].…”

Plant Molecular Farming: Much More than Medicines

“…Scheller et al (82) were the first to produce spider silk proteins in plants, achieving yields of up to 2% TSP in tobacco leaves and potato tubers. Various strategies have been developed to ensure that recombinant silk proteins are produced at the correct native size, including dimerization by nonrepetitive C-termini, cross-linking by transglutaminase, and inteinmediated multimerization (16,83,84).…”

With great structure comes great functionality: Understanding and emulating spider silk

“…52 Recent work using transgenic expression of ELPylated spider silk proteins in tobacco, however, appears to have overcome the problem of low molecular weight expression. 92 After purifying the fusion proteins by inverse transition cycling and crosslinking with transglutaminase, high molecular weight protein films could be produced.…”