Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Plasmids
2.2. Crude Extract Preparation
2.3. Preparing Linear DNA Template for Cell-Free Colicin Production
2.4. Preparing E3 Immunity Protein
2.5. CFPS Reaction
2.6. Quantifying Colicins Using Radioactive 14C-Leu Assay
2.7. Cell Viability Test
2.8. Statistical Analysis
3. Results and Discussion
3.1. Enrichment of Cell Extracts with Chaperones Does Not Significantly Affect CFPS Productivity
3.2. Solubility of Colicin M is Increased in the Presence of Chaperones in CFPS
3.3. Increased Colicin M Solubility Enhances Cell-Killing Activity
3.4. Co-Expression of Colicin E3 and Its Immunity Protein Enhances E3 Activity
3.5. Colicin E1 Is Rapidly Produced and Remains Stable in CFPS
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Strains and Plasmids | Genotype/Relevant Characteristics | Source |
---|---|---|
Strains | ||
E. coli K361 | Wild type W3110 strain with StrR | [30] |
E. coli BL21 Star (DE3) | F− ompT, hsdSB (rB−mB−), gal, dcm, rne131 (DE3) | Invitrogen |
E. coli TG1 | Strain containing colicin plasmid | [32] |
Plasmids | ||
pJL1-sfGFP | KmR, PT7::sfGFP, C-terminal Strep-tag | [33] |
pJL1-cma | KmR, PT7::cma encoding colicin M | [9] |
pJL1-E3 imm | KmR, PT7::E3imm encoding E3 immunity | This study |
pKSJ331 | AmR, ColE1 operon | [32] |
pKSJ167 | AmR, ColE3 operon | [34] |
pGro7 | CmR, ParaB::groES-groEL encoding GroES/EL | Takara Bio |
pKJE7 | CmR, ParaB::dnaK-dnaJ-grpE encoding DnaK/DnaJ/GrpE | Takara Bio |
pG-KJE8 | CmR, ParaB::dnaK-dnaJ-grpE encoding DnaK/DnaJ/GrpE, PPzt-1::groES-groEL encoding GroES/EL | Takara Bio |
Extracts | Solubility (%) | |
---|---|---|
30 °C | RT | |
Star | 16 ± 4 | 16 ± 3 |
Gro | 27 ± 2 | 30 ± 2 |
KJE | 86 ± 3 | 80 ± 3 |
Gro-KJE | 104 ± 2 | 102 ± 9 |
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Jin, X.; Kightlinger, W.; Hong, S.H. Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins. Methods Protoc. 2019, 2, 28. https://doi.org/10.3390/mps2020028
Jin X, Kightlinger W, Hong SH. Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins. Methods and Protocols. 2019; 2(2):28. https://doi.org/10.3390/mps2020028
Chicago/Turabian StyleJin, Xing, Weston Kightlinger, and Seok Hoon Hong. 2019. "Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins" Methods and Protocols 2, no. 2: 28. https://doi.org/10.3390/mps2020028