Protein Expression and Processing

Background

Abstract

Following genetic design of our constructs, we must express and process them into functional materials. Here, we highlight the methods that we used to take our silks from DNA to proteins and ultimately to fibers and films.

Introduction

To create functionalized fibers, we co-spun the NCSilkGFP with native Bombyx mori silk. The NCSilkGFP was designed such that the N and C termini have affinity to b.mori silk, allowing it to bind to the native silk upon spinning.

Methods for co-spinning silk with the NCSilkGFP

In brief, we followed the standard process as highlighted in the literature ^[1] to produce a concentrated aqueous solution, or dope, of commercially purchased b.mori silk. We then added aqueous NCSilkGFP to the concentrated b.mori dope so that the final solution had 750 grams of b.mori silk to 1 gram of NCSilkGFP. We then extruded this dope into a coagulation bath of 90% v/v isopropanol and water and collected the resulting fiber on a godet. Detailed steps are as follows:

Degumming

Silk is comprised of two main proteins, fibroin and sericin. Fibroin is the structural protein of the silk and our protein of interest. Sericin serves as the ‘glue’ of the silk. Degumming separates and removes sericin and is an essential preparation step before dissolving the silk. We found that commercially degummed silk was not properly degummed for our purposes and this step had to be carried out in-lab.

To degum, we boiled 2.5 grams of B.mori silk in 0.02M sodium carbonate solution for 30 minutes.

Solubilization

The resilience of silk to many different solvents is well known. In order to dissolve silk, we used lithium bromide, a strong chaotrope that disrupts the hydrogen bonds and thus secondary structure of the silk.

We dissolved our silk in 9.3M LiBr at 60 C for 4 hours.

Deionizing Dialysis

Following the dissolution of silk, we dialyzed the solution to remove the LiBr and any other salts to obtain a purely aqueous solution of silk.

We dialyzed our silk in SnakeSkin Dialysis Tubing of 3.5 kDa molecular weight cutoff against double deionized water. We dialyzed for 48 hours with a total of 6 dialysis bath changes. Following dialysis, we centrifuged the solution to remove any flocculents and other insoluble proteins.

Concentration Dialysis

The previous dialysis results in an aqueous silk solution with concentration ranging from 4% w/v to 8% w/v (40 - 80 mg/mL). This is too dilute to spin into a fiber, so the solution must be concentrated by dialyzing against a highly concentrated polymer solution. The polymer solution draws water out from the silk solution by osmotic pressure.

To concentrate, we dialyzed 10 mL of aqueous silk solution in a Slide-A-Lyzer dialysis cassette of 3.5 kDa Molecular weight cutoff against a solution of 10% w/v 10,000 molecular weight PEG for 18 hours. This will yield an aqueous silk solution of 15-18% w/v (150-200 mg/mL) concentration. Dialyzing the solution for too long will overconcentrate the silk dope, which will lead to the silk forming a gel in the cassette. Once a silk has gelled, it is no longer usable in any further processing steps.

Spiking with the NCSilkGFP

NCSilkGFP was added to the silk dope such that the resulting solution had a 1:750 mass to mass ratio of NCSilkGFP to native B.mori silk.

Spinning the composite silk

In order to form fibers from silk, soluble silk protein solutions must be much like how they are in natural spider spinnerets. The majority of spinning methods entail pushing, or extruding, silk solution through very thin channels. During this extrusion, shear forces on the silk solution cause the amino acids of the proteins to align in a way that allows the strong beta sheets of the silk structure to form. Multiple proteins are similarly aligned, causing separate proteins to interact and form larger structures.

We used a 21 gauge needle to load the silk dope into a 1 mL BD syringe with luer lok. We then replaced the needle with PEEK tubing of 0.127 mm inner diameter. We used a syringe pump to extrude the silk at a rate of 10 uL/min into a coagulation bath of 90% v/v isopropanol and water. After the fiber formed, we drew it out of the bath and wound it around a motorized godet to collect the fiber as it formed.

Measuring fiber diameter and fluorescence

We used an EVOS light microscope to image the fibers under white light and blue excitation light. We used ImageJ to determine fluorescence of the fiber.

Testing the composite silk

We tested our co-spun fibers in an Instron model 5564 table mounted system with a 10kN load cell. To prepare our fibers for testing, we mounted them on cardstock frames with 10 mm gauge length. We then loaded the carded fibers into the Instron with pneumatic grips and cut the sides of the cardstock frame. We then had the machine extend the fiber at a rate of 0.1 mm/min and measure the tensile force of the fiber.