Log In Sign up
The largest database of trusted experimental protocols

E coli rosetta cells

Manufactured by Merck Group
Visit Supplier
Sourced in Germany

E. coli Rosetta cells are a specialized strain of Escherichia coli bacteria designed for recombinant protein expression. They are engineered to enhance the expression of proteins that require rare tRNAs found in E. coli. The Rosetta cells provide tRNAs that are rarely found in standard E. coli strains, allowing for improved translation of heterologous proteins.

Automatically generated - may contain errors

Lab products found in correlation

Ni sepharose resin, by Cytiva (2 mentions) Source 15q column, by Cytiva (2 mentions) Factor xa, by New England Biolabs (1 mentions) Monos column, by GE Healthcare (1 mentions) Ni nta agarose bead, by Qiagen (1 mentions) Quickchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)

Close spelling variants of this product

E. coli Rosetta 2(DE3) Singles Competent Cells E. coli Rosetta 2(DE3) cells E. coli Rosetta 2(DE3) pLysS cells E. coli BL21 (DE3) Rosetta 2 cells Rosetta 2(DE3) competent E. coli cells E. coli Rosetta 2 cells

5 protocols using e coli rosetta cells

1

Purification of Ricin A-chain Variants

Check if the same lab product or an alternative is used in the 5 most similar protocols
RTA His-tag, modified RTADHF His-tag and RTAIHF His-tag were expressed in E. coli Rosetta cells (Merck) and purified using Ni-NTA agarose beads (Qiagen, Germantown, MD) according to the manufacturer’s manual. The eluate was finally dialysed overnight in PBS.
Ricin A-chain sulf-1 and modified RTAIHF and RTADHF sulf-1 fused to maltose binding protein (MBP) were applied to a column with amylose resin and purified as previously described [22 (link)]. Free wild-type RTA, RTADHF or RTAIHF were cleaved off with factor Xa (New England Biolabs, Ipswich, MA). For further purification, wild-type RTA, RTADHF or RTAIHF proteins were applied on a MonoS column (GE Healthcare) and purified using GE Pharmacia Acta Purifier (GE Healthcare). 25 mM phosphate buffer, pH 6.5 was used as the column equilibrating buffer and the wash buffer, proteins were eluted with 0–500 mM NaCl gradient, and the fractions containing ricin A-chain were identified by Coomassie-stained SDS/PAGE. Purified wild-type RTA, RTADHF or RTAIHF were mixed with the ricin B-chain and dialyzed extensively against PBS to remove reducing agents.
Sokołowska I., Piłka E.S., Sandvig K., Węgrzyn G, & Słomińska-Wojewódzka M. (2015). Hydrophobicity of protein determinants influences the recognition of substrates by EDEM1 and EDEM2 in human cells. BMC Cell Biology, 16, 1.
+ Open protocol
+ Expand
2

TCR and pHLA Expression and Purification

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
TCR chains were expressed individually in E. coli Rosetta cells (Novagen/Merck, Germany) as insoluble inclusion bodies and purified largely as described.13 (link) Briefly, TCRs were produced by refolding in 8M urea and slow dialysis. A molar ratio of 1.2:1 alpha to beta chain was mixed under denaturing conditions and dialyzed for up to 48 hours in 10 mM Tris pH 8.0 before performing ion exchange chromatography (HiTrap MonoQ; Cytiva, USA), followed by size-exclusion chromatography (HR 16/600 or HR 10/300 columns; GE Healthcare, UK). pHLA was produced similarly but with a 1:2 molar ratio of HLA alpha chain to β-2-microglobulin and an excess added of the MAGE-A10-GLY 9-mer peptide (>98% purity; Peptide Protein Research, UK) with up to 60 hours of refolding with dialysis, followed by ion exchange chromatography and size-exclusion chromatography. TCR–pHLA complexes were formed by mixing equimolar quantities of pHLA and TCR, and final purification by size-exclusion chromatography in phosphate-buffered saline.
Simister P.C., Border E.C., Vieira J.F, & Pumphrey N.J. (2022). Structural insights into engineering a T-cell receptor targeting MAGE-A10 with higher affinity and specificity for cancer immunotherapy. Journal for Immunotherapy of Cancer, 10(7), e004600.
+ Open protocol
+ Expand
3

Recombinant Bmr6 Protein Expression and Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols
The Bmr6 coding region was PCR amplified from cDNA using the primers Bmr6F: 5'gatctgggtaccatggggagcctggcgtccgagagggga-3' and Bmr6R: 5'-cgcaagctttcagttgctcggcgcatcagcgg-3'and the product was ligated into a pET30a cloning vector (Novagen, Madison, WI) as a KpnI-HindIII fragment (Sattler et al 2009) . The point mutations identified in the novel bmr6 alleles were introduced into the coding region through site directed mutagenesis using the QuickChange Site Directed Mutagenesis Kit (Agilent, Santa Clara, CA), and validated through commercial DNA sequencing (Operon, Huntsville, AL). Recombinant vectors were introduced into competent E. coli Rosetta cells (Novagen, EMD Millipore, Billerica, MA). Cultures derived from a single colony were grown to log phase at 37 °C and protein expression was induced by exposure to 0.1 M isopropyl β-D-1-thiogalactopyranoside. Induced cells were incubated for 18 hours at 20 °C. Soluble proteins were extracted from transformed cells via sonication at 20 W for three minutes alternating between a 10 second pulse followed by a 30 second rest period.
The expressed recombinant proteins containing N-terminal His tags were purified via affinity nickel column chromatography (GE Healthcare, Little Chalfont, United Kingdom) and were eluted with 0.5M imidazole. Protein induction was confirmed by SDS-PAGE.
Scully E.D., Gries T., Funnell-Harris D.L., Xin Z., Kovacs F.A., Vermerris W, & Sattler S.E. (2016). Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum. Journal of integrative plant biology, 58(2).
+ Open protocol
+ Expand
4

Purification of DDX39B and Variants

Check if the same lab product or an alternative is used in the 5 most similar protocols
All proteins were expressed in E. coli Rosetta cells (Sigma-Aldrich). Protein expression was induced by 0.5 mM IPTG at 20°C overnight. Cells were lysed in a lysis buffer containing 50 mM Tris, pH 8.0, 300 mM NaCl, 0.5 mM TCEP, 1 mM PMSF, and 5 mg/L aprotinin. The GST-tagged proteins were pulled down using Glutathione Sepharose 4B resin and the His-tagged DDX39B was pulled down using Ni Sepharose resin (Cytiva). GST-tagged DDX39B-CTD variants were digested with GST-TEV overnight and purified on a Source 15Q column (Cytiva). The proteins were then passed over Glutathione Sepharose 4B resin to remove remaining GST and uncleaved protein. Purified DDX39B-CTD variants were concentrated, aliquoted, and stored at −80°C in 10 mM Tris, pH 8.0, 300 mM NaCl, 0.5 mM TCEP, and 10% glycerol. The other affinity-purified proteins were first purified on a mono Q column and were subjected to overnight digestion with GST-TEV (for GST-tagged proteins) or His-TEV (for His-tagged DDX39B) to remove the affinity tag. The digested proteins were passed over Glutathione Sepharose 4B resin or Ni Sepharose resin to remove uncleaved protein and TEV. The proteins were further purified on a Superdex 200 column equilibrated with 10 mM Tris, pH 8.0, 150 mM NaCl, and 0.5 mM TCEP. All purified proteins were concentrated, aliquoted, flash frozen in liquid nitrogen, and stored at −80°C.
Xie Y., Gao S., Zhang K., Bhat P., Clarke B.P., Batten K., Mei M., Gazzara M., Shay J.W., Lynch K.W., Angelos A.E., Hill P.S., Ivey A.L., Fontoura B.M, & Ren Y. (2023). Structural basis for high-order complex of SARNP and DDX39B to facilitate mRNP assembly. Cell reports, 42(8), 112988.
+ Open protocol
+ Expand
5

Purification of DDX39B and Variants

Check if the same lab product or an alternative is used in the 5 most similar protocols
All proteins were expressed in E. coli Rosetta cells (Sigma-Aldrich). Protein expression was induced by 0.5 mM IPTG at 20°C overnight. Cells were lysed in a lysis buffer containing 50 mM Tris, pH 8.0, 300 mM NaCl, 0.5 mM TCEP, 1 mM PMSF, and 5 mg/L aprotinin. The GST-tagged proteins were pulled down using Glutathione Sepharose 4B resin and the His-tagged DDX39B was pulled down using Ni Sepharose resin (Cytiva). GST-tagged DDX39B-CTD variants were digested with GST-TEV overnight and purified on a Source 15Q column (Cytiva). The proteins were then passed over Glutathione Sepharose 4B resin to remove remaining GST and uncleaved protein. Purified DDX39B-CTD variants were concentrated, aliquoted, and stored at −80°C in 10 mM Tris, pH 8.0, 300 mM NaCl, 0.5 mM TCEP, and 10% glycerol. The other affinity-purified proteins were first purified on a mono Q column and were subjected to overnight digestion with GST-TEV (for GST-tagged proteins) or His-TEV (for His-tagged DDX39B) to remove the affinity tag. The digested proteins were passed over Glutathione Sepharose 4B resin or Ni Sepharose resin to remove uncleaved protein and TEV. The proteins were further purified on a Superdex 200 column equilibrated with 10 mM Tris, pH 8.0, 150 mM NaCl, and 0.5 mM TCEP. All purified proteins were concentrated, aliquoted, flash frozen in liquid nitrogen, and stored at −80°C.
Xie Y., Gao S., Zhang K., Bhat P., Clarke B.P., Batten K., Mei M., Gazzara M., Shay J.W., Lynch K.W., Angelos A.E., Hill P.S., Ivey A.L., Fontoura B.M, & Ren Y. (2023). Structural basis for high-order complex of SARNP and DDX39B to facilitate mRNP assembly. Cell reports, 42(8), 112988.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!