|Name:||LOBSTR E. coli Expression Strain|
|Cell Type:||Chemically competent (CaCl2method)|
|Organism:||E. coli BL21(DE3)|
|Growth Conditions:||Standard E.coli Growth Media (LB, SOC, etc.) at 37C|
|Transformation:||Standard heatshock protocol (42C for 20 seconds)|
|Induction:||IPTG up to 1mM|
|Comments:||Derived from strain YYC202, strain has a growth requirement for acetate and isoleucine|
|Storage:||-80C (avoid freeze-thaw cycles)|
LOBSTR and the parental BL21(DE3) strain show comparable growth
The growth (OD600) of both LOBSTR and the parental BL21(DE3) strain was measured from initial synchronization at 0 h until the final harvest. Both strains carried the same expression plasmid and were grown at 37C until an OD600 ~0.7, at which point protein expression was induced at 18, 25, and 37C (black arrow). The growth curves for LOBSTR and BL21(DE3) are shown in red and black, respectively. Cell growth during log phase and final cell density was similar for both strains. Depending on the expression plasmid different growth behavior was observed, but typically no growth difference was seen between LOBSTR and BL21(DE3).
ArnA and SlyD are eliminated from His-tag purifications from LOBSTR
Elution samples of test purifications from BL21(DE3) and LOBSTR using common metal affinity resins are shown. (A) Seven protein constructs were purified from both the parental BL21(DE3) strain and LOBSTR using Ni Sepharose 6FF resin (GE Healthcare). The constructs are numbered 1-7, and contain either a 6×His-tag (1 and 4) or a 10×His-tag (2,3,57). The elution samples were run on an SDS-PAGE gel and stained with Coomassie Blue R250. ArnA and SlyD are indicated by arrows and target proteins indicated with a black circle (). The double asterisk (**) indicates Hsp15, another protein showing reduced Ni-binding affinity in LOBSTR. (B) Purifications of constructs 1 and 5 from BL21(DE3) and LOBSTR were also carried out on two additional commonly used resins, Ni-NTA (Qiagen) and Talon (Clontech). In each case, ArnA and SlyD are successfully eliminated in LOBSTR.
Adapted from: Andersen KR, et al. Proteins. 2013 Nov;81(11):1857-61.
LOBSTR E. Coli strain characterization
- Andersen KR, Leksa NC, Schwartz TU. Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins. 2013 Nov;81(11):1857-61.
LOBSTR E. Coli strain utilization
- Saxton RA, Knockenhauer KE, Wolfson RL, Chantranupong L, Pacold ME, Wang T, Schwartz TU, Sabatini DM. Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway. Science. 2016 Jan 1;351(6268):53-8. doi: 10.1126/science.aad2087. View Article
- Saxton RA, Chantranupong L, Knockenhauer KE, Schwartz TU, Sabatini DM. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. Nature. 2016 Aug 11;536(7615):229-33. View Article
- Huhn AJ, Guerra RM, Harvey EP, Bird GH, Walensky LD. Selective CovalentTargeting of Anti-Apoptotic BFL-1 by Cysteine-Reactive Stapled PeptideInhibitors. Cell Chem Biol. 2016 Sep 7. pii: S2451-9456(16)30289-6. View Article
- Demircioglu FE, Sosa BA, Ingram J, Ploegh HL, Schwartz TU. Structures ofTorsinA and its disease-mutant complexed with an activator reveal the molecularbasis for primary dystonia. Elife. 2016 Aug 4;5. pii: e17983. View Article
- Truttmann MC, Cruz VE, Guo X, Engert C, Schwartz TU, Ploegh HL. The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones. PLoS Genet. 2016 May 3;12(5):e1006023. View Article
- Knockenhauer KE, Schwartz TU. Structural Characterization of Bardet-Biedl Syndrome 9 Protein (BBS9). J Biol Chem. 2015 Jun 17. pii: jbc.M115.649202.
- Kelley K, Knockenhauer KE, Kabachinski G, Schwartz TU. Atomic structure of the Y complex of the nuclear pore. Nat Struct Mol Biol. 2015 May;22(5):425-31. doi: 10.1038/nsmb.2998. Epub 2015 Mar 30.
- Sosa BA, Demircioglu FE, Chen JZ, Ingram J, Ploegh HL, Schwartz TU. How lamina-associated polypeptide 1 (LAP1) activates Torsin. Elife. 2014 Aug 22;3:e03239. doi: 10.7554/eLife.03239.
- Saxton RA, Knockenhauer KE, Schwartz TU, Sabatini DM. The apo-structure of the leucine sensor Sestrin2 is still elusive. Sci Signal. 2016 Sep 20;9(446):ra92. doi: 10.1126/scisignal.aah4497. PubMed PMID: 27649739; PubMed Central PMCID: PMC5087270. View Article
- Saxton RA, Chantranupong L, Knockenhauer KE, Schwartz TU, Sabatini DM. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. Nature. 2016 Aug 11;536(7615):229-33. PubMed PMID: 27487210; PubMed Central PMCID: PMC4988899. View Article
- Kawaharada Y, Nielsen MW, Kelly S, James EK, Andersen KR, Rasmussen SR, Füchtbauer W, Madsen LH, Heckmann AB, Radutoiu S, Stougaard J. Differential regulation of the Epr3 receptor coordinates membrane-restricted rhizobial colonization of root nodule primordia. Nat Commun. 2017 Feb 23;8:14534. doi: 10.1038/ncomms14534. PubMed PMID: 28230048; PubMed Central PMCID: PMC5331223. View Article
- Sander B, Xu W, Eilers M, Popov N, Lorenz S. A conformational switch regulates the ubiquitin ligase HUWE1. Elife. 2017 Feb 14;6. pii: e21036. doi: 10.7554/eLife.21036. PubMed PMID: 28193319; PubMed Central PMCID: PMC5308896. View Article
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