Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae
Abstract
:1. Introduction
2. Materials and Methods
2.1. Protein Clustering
2.2. Esterase Activity
2.3. Gene Cloning, Expression, and Purification of Recombinant LgEstI Protein
2.4. LgEstI Crystallization, Data Collection, and Phasing
3. Results
3.1. Clustering Analysis of LgEstI
3.2. Purification and Biochemical Characterization of LgEstI
3.3. X-ray Crystallographic Study of LgEstI
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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LgEst | |
---|---|
Source organism | Lactococcus garvieae |
DNA source | Genomic DNA |
Cloning vector | pET21a |
Expression host | Escherichia coli BL21 (DE3) |
Amino acid sequence | MVERISLEKAALEFSEANAPHPRIYELPVEEGRSLLNEV QDSPVVKEDVDIEDIAVDTGEWGEINVRFIRPLHQEKKL PVIFYIHGAGWVFGNAHTHDKLIRELAVRTNSVVVFSE YSLSPEAKYPTAIEQNYAVLQQLKDFANDKKFDVNHLT VAGDSVGGNMATVMTLLTKQRGGQKIGQQVLYYPVT DANFDTDSYNEFAENYFLTKEGMIWFWDQYTTSQEER HQITASPLRATKEDLADLPAALIITGEADVLRDEGEAYA RKLREADVEVTQVRFQAIIHDFVMVNSMNETHATRAA MSLSTQWINEKNRK |
Method | Vapor Diffusion |
---|---|
Plate type for screening | 96-well sitting drop MRC plate (Molecular Dimension, Suffolk, UK) |
Plate type for optimization | 24-well hanging drop plate (Molecular Dimension, Suffolk, UK) |
Temperature (°C) | 22 |
Protein concentration (mg/mL) | 25 |
Composition of protein solution | 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 1 mM TECP |
Composition of reservoir solution | 0.1 M Tris: HCl (pH 7.1), 0.2 M calcium acetate hydrate, and 19% (w/v) PEG 3000 |
Volume and ratio of drop | 2 μL, 1:1 |
Volume of reservoir (μL) | 500 |
Data Collection | LgEstI |
---|---|
Wavelength (Å) | 0.97949 |
X-ray source | BL-5C beamline |
Rotation range per image (°) | 1 |
Exposure time (s) | 0.1 |
Space group | I21 |
Unit-cell parameters (Å, °) | a = 54.41, b = 92.77, c = 218.11, α = γ = 90.0, β = 96.61 |
Resolution range (Å) a | 28.49–2.0 (2.05–2.0) |
No. of observed reflections a | 494,404 |
No. of unique reflections a | 71,847 (4652) |
Completeness (%) a | 98.7 (99.8) |
Redundancy a | 6.9 (7.2) |
Rsyma,b | 0.059 (0.540) |
I/σ a | 16.1 (2.8) |
Mosaicity | 0.36 |
CC½ a,c | (99.7%) |
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Do, H.; Wang, Y.; Lee, C.W.; Yoo, W.; Jeon, S.; Hwang, J.; Lee, M.J.; Kim, K.K.; Kim, H.-W.; Lee, J.H.; et al. Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae. Crystals 2022, 12, 46. https://doi.org/10.3390/cryst12010046
Do H, Wang Y, Lee CW, Yoo W, Jeon S, Hwang J, Lee MJ, Kim KK, Kim H-W, Lee JH, et al. Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae. Crystals. 2022; 12(1):46. https://doi.org/10.3390/cryst12010046
Chicago/Turabian StyleDo, Hackwon, Ying Wang, Chang Woo Lee, Wanki Yoo, Sangeun Jeon, Jisub Hwang, Min Ju Lee, Kyeong Kyu Kim, Han-Woo Kim, Jun Hyuck Lee, and et al. 2022. "Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae" Crystals 12, no. 1: 46. https://doi.org/10.3390/cryst12010046