Improved Production and Biophysical Analysis of Recombinant Silicatein-α
Abstract
:1. Introduction
2. Materials and Methods
2.1. Gene Cloning and Protein Overexpression
2.2. Circular Dichroism
2.3. Dynamic Light Scattering, Static Light Scattering and Intrinsic Fluorescence
2.4. Enzyme Activity Assays
3. Results and Discussion
3.1. Design of Recombinant Protein Constructs
3.2. Optimisation of Silα Purification
3.3. Circular Dichroism Spectroscopy
3.4. Dynamic Light Scattering
3.5. Temperature-Dependent Melting
3.6. Temperature-Dependent Aggregation
3.7. Enzymatic Activity
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Entry | Buffer a | Additives b | pH | Presence of Soluble Protein After Lysis c | IMAC Compatible d |
---|---|---|---|---|---|
1 | Tris | NaCl, L-Arg, L-Glu | 8.5 | × | n.d. |
2 | EPPS | NaCl, L-Arg, L-Glu | 8.5 | × | n.d. |
3 | BisTris | NaCl, L-Arg, L-Glu | 8.5 | × | n.d. |
4 | CHES | NaCl, L-Arg, L-Glu | 9.5 | √ | × |
5 | Tris | NaCl, CHAPS (5 mM), Triton X-100 (1%) | 8.5 | √ | × |
6 | CHES | NaCl | 9.5 | √ | × |
7 e | Tris | NaCl | 7.5 | × | n.d. |
8 | Tris | NaCl | 8.5 | √ | √ |
9 | AMP | NaCl | 8.5 | √ | × |
10 | AMP | NaCl | 9.0 | √ | × |
11 | AMP | NaCl | 9.5 | √ | × |
12 | AMP | NaCl | 10.0 | √ | × |
13 | AMP | NaCl | 10.5 | √ | × |
Entry | Buffer a | Additives a | pH | Presence of Soluble Protein After Lysis b | SAC Compatible c |
---|---|---|---|---|---|
1 | CHES | NaCl, L-Arg, L-Glu | 9.5 | √ | √ |
2 | Tris | NaCl, CHAPS (5 mM), Triton X-100 (1%) | 8.5 | √ | √ |
3 | CHES | NaCl | 9.5 | √ | √ |
4 | Tris | NaCl | 8.5 | √ | √ |
5 | AMP | NaCl | 8.5 | √ | √ |
6 | AMP | NaCl | 9.0 | √ | √ |
7 | AMP | NaCl | 9.5 | √ | √ |
8 | AMP | NaCl | 10.0 | √ | √ |
9 | AMP | NaCl | 10.5 | √ | √ |
Buffer | Additives | pH |
---|---|---|
Potassium phosphate (100 mM) | KCl (20 mM) | 7.0 |
Tris-HCl (50 mM) | NaCl (100 mM) | 8.5 |
AMP (50 mM) | NaCl (100 mM) | 9.0 |
Protein | Buffer | pH | Isolated Yield (nmol L−1 of Cell Culture) a |
---|---|---|---|
His6-Silα | Tris (50 mM), NaCl (100 mM) | 8.5 | 8 |
His6-TF- Silα | Potassium phosphate (100 mM) | 8.0 | 6 |
Tris (50 mM), NaCl (100 mM) | 8.5 | 177 | |
TF-Silα-Strep | Tris (50 mM), NaCl (100 mM) | 8.5 | 777 |
AMP (50 mM), NaCl (100 mM) | 9.0 | 1540 |
pH | His6-TF-Silα | TF-Silα-Strep | ||
---|---|---|---|---|
% Polydispersity | PDI | % Polydispersity | PDI | |
7.0 | 93.8 ± 27.3 | 1.65 ± 0.57 | 41.6 ± 6.4 | 0.326 ± 0.047 |
8.5 | 130.3 ± 15.6 | 2.29 ± 0.07 | 49.3 ± 1.7 | 0.302 ± 0.062 |
9.0 | 61.7 ± 21.5 | 1.47 ± 0.68 | 34.5 ± 6.7 | 0.152 ± 0.079 |
Enzyme | KM (µM) | kcat (min−1) | kcat/KM (min−1 µM−1) |
---|---|---|---|
His6-TF-Silα | 22.4 ± 2.2 a | 988 ± 416 a | 44.1 |
TF-Silα-Strep | 88.2 ± 34.6 | 0.150 ± 0.033 | 0.00170 |
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Sparkes, E.I.; Kettles, R.A.; Egedeuzu, C.S.; Stephenson, N.L.; Caslin, S.A.; Tabatabaei Dakhili, S.Y.; Wong, L.S. Improved Production and Biophysical Analysis of Recombinant Silicatein-α. Biomolecules 2020, 10, 1209. https://doi.org/10.3390/biom10091209
Sparkes EI, Kettles RA, Egedeuzu CS, Stephenson NL, Caslin SA, Tabatabaei Dakhili SY, Wong LS. Improved Production and Biophysical Analysis of Recombinant Silicatein-α. Biomolecules. 2020; 10(9):1209. https://doi.org/10.3390/biom10091209
Chicago/Turabian StyleSparkes, Emily I., Rachel A. Kettles, Chisom S. Egedeuzu, Natalie L. Stephenson, Stephanie A. Caslin, S. Yasin Tabatabaei Dakhili, and Lu Shin Wong. 2020. "Improved Production and Biophysical Analysis of Recombinant Silicatein-α" Biomolecules 10, no. 9: 1209. https://doi.org/10.3390/biom10091209
APA StyleSparkes, E. I., Kettles, R. A., Egedeuzu, C. S., Stephenson, N. L., Caslin, S. A., Tabatabaei Dakhili, S. Y., & Wong, L. S. (2020). Improved Production and Biophysical Analysis of Recombinant Silicatein-α. Biomolecules, 10(9), 1209. https://doi.org/10.3390/biom10091209