Biochemical and Initial Structural Characterization of the Monocot Chimeric Jacalin OsJAC1
Abstract
:1. Introduction
2. Results
2.1. Heterologous Expression and Isolation of the OsJAC1 Protein from Oryza sativa and Its JRL and DIR Domains
2.2. Properties of the Chimeric Lectin OsJAC1—Initial Structural Characterization
2.2.1. Investigation of Oligomericity
2.2.2. Melting Point (Tm)
2.2.3. Circular Dichroism (CD) Spectroscopy
2.3. Interaction Partners of the Lectin Domain
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Western Blot Using Transgenic Plants Tissue
4.3. Construct and Cloning
4.4. Heterologous Expression of OsJAC1 and Its DIR and JRL Domains
4.5. Isolation of OsJAC1 and the Single Domains (DIR and JRL)
4.6. Protein Concentration Determination
4.7. MALDI-TOF-MS Analysis
4.8. DLS
4.9. Differential Scanning Fluorimetry
4.10. CD Spectroscopy
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
C | control |
CD | circular dichroism |
CFE | cell free extract |
DIR | dirigent protein |
DLS | dynamic light scattering |
DSF | differential scanning fluorimetry |
DTT | dithiothreitol |
F# | SEC fraction |
FT | flow through |
GFP | green fluorescent protein |
GST | glutathione S-transferase-tag |
IMAC | immobilized metal-chelate affinity chromatography |
JRL | jacalin-related lectin |
JRL + DIR | stoichiometric mixture of JRL and DIR domain |
gJRL | galactose-specific jacalin-related lectins |
mJRL | mannose-specific jacalin-related lectins |
M | marker |
OsJAC1_P1 | main OsJAC1 peak of SEC |
OsJAC1_P2 | secondary OsJAC1 peak of SEC |
PMSF | phenylmethylsulfonyl fluoride |
RE | raw extract/disrupted cell suspension |
RT | room temperature |
SEC | size-exclusion chromatography |
Tm | melting point |
W1 | wash fraction with 100% buffer A (20 mm imidazol) |
WF | wash fraction with 30% buffer B (250 mm imidazol) |
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OsJAC1 | DIR | JRL | |
---|---|---|---|
residue range | 1–329 | 28–182 | 183–329 |
molecular mass [kDa] | 35.57 | 19.3 | 18.39 |
plasmid | pColdIV | pET15b(+) | pET15b(+) |
expression condition | 10 °C for 3.5 d | 10 °C for 3.5 d | 10 °C for 3.5 d |
pellet size [g] | 7 | 5.8 | 8.6 |
purity [%] a | >95 | >95 | >95 |
conc. after IMAC [mg mL−1] | 10.7 | 9.7 | 8.1 |
VFinal [mL] | 3.5 | 3.5 | 3.5 |
appearance | light yellow color | light yellow color | colorless |
Buffer Condition | Min. MW/kDa | Max. MW/MDa | No. of Distinct Species |
---|---|---|---|
15 mm TRIS-HCl pH 7.4 | 308 | 26.95 | two |
+150 mm CaCl2 | 268.8 | 3317 | many |
+50 mm lactose | 187.4 | - | one |
+150 mm MgSO4 | 183.7 | - | one |
+4 mm DTT | 137.5 | - | one |
DTT | Sample | Initial ratio a | ∆ ratio b | TM1 (°C) (±) | TM2 (°C) (±) |
---|---|---|---|---|---|
− | JRL | 0.95 | −0.03 | n.d. | 74.3 ± 0.04 |
− | DIR | 0.63 | 0.23 | n.a. | 77.4 ± 0.01 |
− | OsJAC1 | 0.70 | 0.17 | n.d. | 85.1 ± 0.03 |
− | JRL + DIR | 0.76 | 0.12 | 60.1 ± 0.02 | 77.7 ± 0.02 |
+ | JRL | 0.96 | −0.03 | 58.4 ± 0.13 | n.d. |
+ | DIR | 0.56 | 0.28 | n.a. | 76.8 ± 0.03 |
+ | OsJAC1 | 0.70 | 0.16 | 59.1 ± 0.01 | 85.4 ± 0.01 |
+ | JRL + DIR | 0.77 | 0.12 | 59.6 ± 0.51 | 77.3 ± 0.05 |
Protein | α-Helix | β-Sheet | β-Turns | Other | Reference |
---|---|---|---|---|---|
OsJAC1 JRL domain | 0% | 46% | 12% | 42% | - |
gJRL [18] | 2% | 34% | 23% | 39% | [18] |
mJRL | 0% | 73% 1 | - | 27% | pdb:4PIF [19] |
OsJAC1 DIR domain | 1% | 47% | 9% | 44% | - |
Dirigent protein GePTS1 | 4% | 56% 1 | - | 40% | pdb:6OOC [20] |
Dirigent protein AtDIR6 | 0% | 56% 1 | - | 44% | pdb:5LAL [21] |
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Huwa, N.; Weiergräber, O.H.; Kirsch, C.; Schaffrath, U.; Classen, T. Biochemical and Initial Structural Characterization of the Monocot Chimeric Jacalin OsJAC1. Int. J. Mol. Sci. 2021, 22, 5639. https://doi.org/10.3390/ijms22115639
Huwa N, Weiergräber OH, Kirsch C, Schaffrath U, Classen T. Biochemical and Initial Structural Characterization of the Monocot Chimeric Jacalin OsJAC1. International Journal of Molecular Sciences. 2021; 22(11):5639. https://doi.org/10.3390/ijms22115639
Chicago/Turabian StyleHuwa, Nikolai, Oliver H. Weiergräber, Christian Kirsch, Ulrich Schaffrath, and Thomas Classen. 2021. "Biochemical and Initial Structural Characterization of the Monocot Chimeric Jacalin OsJAC1" International Journal of Molecular Sciences 22, no. 11: 5639. https://doi.org/10.3390/ijms22115639
APA StyleHuwa, N., Weiergräber, O. H., Kirsch, C., Schaffrath, U., & Classen, T. (2021). Biochemical and Initial Structural Characterization of the Monocot Chimeric Jacalin OsJAC1. International Journal of Molecular Sciences, 22(11), 5639. https://doi.org/10.3390/ijms22115639