Protein Engineering of Multi-Modular Transcription Factor Alcohol Dehydrogenase Repressor 1 (Adr1p), a Tool for Dissecting In Vitro Transcription Activation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Design of the Minimal Regulatory Units
2.2. Cloning and Expression
2.2.1. Cloning
2.2.2. Expression in Escherichia coli
2.2.3. Expression in Komagataella pastoris
2.3. Purification of miniAdr1 Constructs
2.3.1. Purification of His-tagged miniAdr1 Constructs
2.3.2. Purification of Untagged miniAdr1 Constructs
2.4. Functional Characterization
2.4.1. DNA-Binding Assays
2.4.2. Quaternary Assembly by HPLC
3. Results
3.1. Design of the Minimal Regulatory Units
3.2. Cloning, Expression, and Purification of miniARD1 Constructs in Escherichia coli and Komagtaella pastoris
3.3. Functional Characterization of miniAdr1 Constructs
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Escherichia coli (His-tag) | Komagataella pastoris (His-tag) | K. pastoris (no tag) | |
---|---|---|---|
Adr1-651 | Insoluble (inclusion bodies, IB) | - | - |
Adr1-651_Δ158–400 | Insoluble (IB) | - | - |
Adr1-471 | Insoluble (IB) | insoluble | - |
MiniAdr1-AL | <1 mg/L culture | <1 mg/L | 1 mg/L unstable1 |
MiniAdr1-BL | <1 mg/L | <1 mg/L | 3 mg/L |
Adr1-DBD | - | - | 1 mg/L unstable1 |
HisAdr1-165 | ~1 mg/L unstable 1 | - | - |
Oligonucleotide Name | Sequence |
---|---|
Palindromic | 5′–CCGGCCTCTCCAACTTA·TAAGTTGGAGAGG–3′ |
Half site “a” | 5′–TCCGGGCATCTCCAACTTA–3′ |
Half site “b” | 5′–CTAAGTTGGAGATGCCCGG–3′ |
Composition of the Translated Proteins with Respect to the Full Length Primary Structure | Linker Sequence | |
---|---|---|
miniAdr1-AL | (75–157)–(401–471) | 401–SWTVAIDNNSNNNKVSDN–418 |
miniAdr1-BL | (75–178)–(419–471) | 158–NLGETISHTKKVSRTITKAR–178 |
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Buttinelli, M.; Panetta, G.; Bucci, A.; Frascaria, D.; Morea, V.; Miele, A.E. Protein Engineering of Multi-Modular Transcription Factor Alcohol Dehydrogenase Repressor 1 (Adr1p), a Tool for Dissecting In Vitro Transcription Activation. Biomolecules 2019, 9, 497. https://doi.org/10.3390/biom9090497
Buttinelli M, Panetta G, Bucci A, Frascaria D, Morea V, Miele AE. Protein Engineering of Multi-Modular Transcription Factor Alcohol Dehydrogenase Repressor 1 (Adr1p), a Tool for Dissecting In Vitro Transcription Activation. Biomolecules. 2019; 9(9):497. https://doi.org/10.3390/biom9090497
Chicago/Turabian StyleButtinelli, Memmo, Gianna Panetta, Ambra Bucci, Daniele Frascaria, Veronica Morea, and Adriana Erica Miele. 2019. "Protein Engineering of Multi-Modular Transcription Factor Alcohol Dehydrogenase Repressor 1 (Adr1p), a Tool for Dissecting In Vitro Transcription Activation" Biomolecules 9, no. 9: 497. https://doi.org/10.3390/biom9090497