NM architecture and recombinant expression
The detailed amino acid sequence of NM is shown in Figure 1A, while Figure 1B presents the architecture. The NM recombinant expression was conducted at 37°C and the final IPTG concentration was 1 mmol/L. Indicated by the SDS-PAGE (Fig. 1C), compared to the control sample (lane U) there is an additional strong band (red arrow) after IPTG induction (lane I), which fits very well to the target protein NM judged by its apparent size of 38 kDa. After bacterial cell disruption, the soluble (lane S) and insoluble cell fractions (lane P) were separated by centrifuge. The NM was mainly in pellet fraction as inclusion bodies (green arrow). These results indicate that recombinant spider silk protein NM can be efficient expressed as inclusion bodies (NM IBs) in E. coli.
Isolation and solubilization of NM IBs
Pure NM IBs were prepared through extensive washing with detergent containing buffer, after which majority of contaminants were removed. The NM IBs purity was evaluated by SDS-PAGE, which is up to 70% (black arrow, Fig. 2A). The purified NM IBs (10 mg/mL wet weight concentration) were resuspended in Tris-HCl pH 8.0 containing different concentrations of urea (0-7 mol/L), and further proceeded with one step-heating method and traditional urea-denature method. The solubilized supernatants of NM IBs from both methods were analyzed by SDS-PAGE (Fig. 2B and C), and the protein concentrations were measured by Micro BCA Protein Assay kit (Table 1), respectively. With traditional urea-denatured method, NM IBs were largely solubilized in Tris-HCl pH 8.0 containing 5–7 mol/L urea, whereas with 0–4 mol/L urea the solubilization efficiency was rather low (Fig. 2B). Interestingly, by performing the one-step heating method Tris-HCl pH 8.0 containing 0–7 mol/L urea all solubilized NM IBs, and at 4 mol/L urea concentration the solubilization efficiency was already up to 80%, which is around three times higher than that of traditional urea-denature method. Interestingly, Tris-HCl pH 8.0 alone could also solubilize NM IBs (Lane 0, Fig. 2C) to some extent. These results indicated that short-term heating improves the solubilization of NM IBs, and the efficiency achieved through one-step heating method in the presence of 4 mol/L urea is comparable to that of 7 mol/L urea via traditional urea-denatured method (Fig. 2D). Hence, for testing other potential affecting factors below, the urea concentration was stuck to 4 mol/L.
Solubilization of NM IBs at different temperatures and pHs
In order to optimize the heating temperature that is essential for solubilization of NM IBs when performing one-step heating method, we tested effects from different temperatures, ranging from 40 to 100°C, in 10 mmol/L Tris-HCl pH 8.0 containing 4 mol/L urea. As shown in Figure. 3, the solubilization capability was progressively increased when the heating temperature rose, and the plateau was reached at around 85°C. Considering the thermal stability of spidroins, the heating temperature is optimized as 85°C for following evaluations. Similarly, to find out the most suitable working pH the purified NM IBs were solubilized in 10 mmol/L Tris-HCl at different pHs (pH 5–10) containing 4 mol/L urea. The suspensions were mixed thoroughly and heated at 85°C for 20 min. The soluble fractions containing NM were analyzed by SDS-PAGE. The results showed the solubilization efficiency was also affected by the working pH, with an increasing tendency from pH 5 to 7, while the different between pH 7, 8, 9 and 10 was not very obvious (Fig. 4), suggesting NM IBs solubilization by one-step heating method can be working under broad pH conditions.
Table 1. Solubilization of NM spidroins from IBs with different concentrations of urea through two different methods
Urea conc. (mol/L)
|
Protein concentration (ng/ml)
|
One-step heating method
|
Traditional urea-denatured
|
0
|
483
|
0
|
1
|
952
|
58
|
2
|
1290
|
157
|
3
|
1866
|
311
|
4
|
2335
|
728
|
5
|
2641
|
1368
|
6
|
2786
|
2143
|
7
|
2912
|
2458
|
Solubilization of NM IBs in different buffers
The solubilization of IBs might be influenced by different normally used buffer conditions. To figure this out, five different buffers were tested in this study. The results showed that the buffer conditions did not significantly affect the solubilization efficiency of NM IBs (Fig. 5A), indicated by the bands with similar intensity from 10 mmol/L NaCO3 (pH 8.0), 10 mmol/L K3PO4 (pH 8.0), 10 mmol/L Tris-HCl (pH 8.0) and deionized water (ddH2O), even though a slight decrease in 1×PBS was observed. These results suggest the one-step heating method holds the potential to work under a wide range of biological buffers without significantly decreasing the solubilization capability.
Self-assembly of NM protein into nanoparticles
To evaluate recombinant NM generated by the one-step heating method in a function point of view, the NM nanoparticle self-assembly was induced by salting out with potassium phosphate. As a result, the recombinant NM successfully self-assembled into nanoparticles. Under scanning electron microscope, well-distributed even spherical nanoparticles with diameter around 500 nm were observed (Fig. 6). The NM nanoparticles shared similar morphology as previous reported nanoparticles formed by other recombinant spidroins [2, 9], and might have potential and specific applications as functional biomaterials, e.g. controllable delivery of protein drugs /peptide vaccines.
Identification of urea-induced modification
To identify potential carbamylation from urea, the recombinant NM generated with above protocol was trypsinated and analyzed by mass spectrometry. Due to technique problems or the peptides their own properties, the peptides detected by mass spectrometry could not cover 100% of the full-length sequence; however, both oxidation and carbamylation were already observed (Supplementary Table 1). The cyanate group was detected on the sidechains of both Arg and Lys residues (Fig. 7, Supplementary Table 1). Although there is no evidence in this study to support the N terminal carbamylation of the recombinant NM solubilized with this heating protocol, still we cannot exclude this possibility. For the downstream experiments that are sensitive to carbamylation, the experiments should be designed carefully.