The subunit DBL1-ID2a of VAR2CSA (rVAR2) was recombinantly expressed in Escherichia coli as previously described49 (link). In brief, the FCR3 DBL1-ID2a with a C-terminal V5 tag, penta-His tag, and a split protein tag sequence was inserted into a modified pET15b plasmid (Novagen) and transformed to SHuffle T7 Express Competent E. coli cells (New England Biolabs, C3029H). Following lysis of the cell pellet, rVAR2 expressed in a soluble form was purified by immobilized affinity chromatography (IMAC) followed by size-exclusion chromatography. Purity of the protein was confirmed by SDS page and Western blot, whereas specificity toward ofCS was ensured in ELISA and on cancer cells using flow cytometry.
E coli shuffle t7 express competent cells
Manufactured by New England Biolabs
Sourced in United Kingdom
E. coli SHuffle T7 Express competent cells are a strain of Escherichia coli bacteria engineered to facilitate the expression and proper folding of recombinant proteins. The cells contain a chromosomal copy of the gene encoding the disulfide bond isomerase DsbC, which helps catalyze the formation and rearrangement of disulfide bonds in the cytoplasm, thereby improving the solubility and activity of expressed proteins.
Automatically generated - may contain errors
Lab products found in correlation
One shot top10 chemically competent e coli cells, by Thermo Fisher Scientific (2 mentions)
Du 800 uv visible spectrophotometer, by Beckman Coulter (1 mentions)
Sorvall lynx 4000 centrifuge, by Thermo Fisher Scientific (1 mentions)
Q5 dna polymerase, by New England Biolabs (1 mentions)
Hispur ni nta spin column, by Thermo Fisher Scientific (1 mentions)
7 protocols using e coli shuffle t7 express competent cells
1
Recombinant Expression and Purification of VAR2CSA
2
Plasmid Amplification and Protein Expression
3
Plasmid Amplification and Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One Shot TOP10 chemically competent E. coli cells (Thermofisher Scientific) were used for amplification of plasmid constructs. Protein expression studies were conducted using SHuffle® T7 Express competent E. coli cells (New England Biolabs, Cat No: C3029J).
4
Recombinant Trx and TrxR Expression in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Coding sequences
for Ta-TrxR and Ta-Trx were amplified from T. acidophilum genomic DNA using Q5 DNA polymerase (NEB, Ipswich, MA), and the
respective amplicons were cloned into NdeI and BamHI sites of pTev5, a T7-based expression vector,23 (link) resulting pUL207 and pUL208, respectively. These
plasmids were designed to express recombinant proteins with an NH2-terminal His6-tag, followed by a TEV protease
recognition site. Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB,
Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA). The
resulting strains were grown at 37 °C in Luria Bertani media
containing 100 and 34 μg/mL ampicillin and chloramphenicol,
respectively. For the expression of Ta-Trx, the LB media was supplemented
with DTT at a final concentration of 0.2 mM. When a growing culture
reached an optical density (OD600) of 0.8, as measured
with a DU800 UV–visible spectrophotometer (Beckman Coulter,
Inc., Brea, CA), IPTG was added to a final concentration of 0.4 mM
and the cultivation was continued for additional 5 h at 37 °C
for Ta-TrxR overexpression and for 12 h at 15 °C for Ta-Trx overexpression.
The cells from these cultures were harvested by centrifugation at
10 000g for 10 min at 4 °C using a Sorvall
LYNX 4000 centrifuge (Thermo Fisher Scientific, Waltham, MA), and
the cell pellets were stored at −20 °C until used.
for Ta-TrxR and Ta-Trx were amplified from T. acidophilum genomic DNA using Q5 DNA polymerase (NEB, Ipswich, MA), and the
respective amplicons were cloned into NdeI and BamHI sites of pTev5, a T7-based expression vector,23 (link) resulting pUL207 and pUL208, respectively. These
plasmids were designed to express recombinant proteins with an NH2-terminal His6-tag, followed by a TEV protease
recognition site. Both plasmids were transformed into E. coli SHuffle T7 Express competent cells (NEB,
Ipswich, MA) containing pRIL plasmid (Stratagene, La Jolla, CA). The
resulting strains were grown at 37 °C in Luria Bertani media
containing 100 and 34 μg/mL ampicillin and chloramphenicol,
respectively. For the expression of Ta-Trx, the LB media was supplemented
with DTT at a final concentration of 0.2 mM. When a growing culture
reached an optical density (OD600) of 0.8, as measured
with a DU800 UV–visible spectrophotometer (Beckman Coulter,
Inc., Brea, CA), IPTG was added to a final concentration of 0.4 mM
and the cultivation was continued for additional 5 h at 37 °C
for Ta-TrxR overexpression and for 12 h at 15 °C for Ta-Trx overexpression.
The cells from these cultures were harvested by centrifugation at
10 000g for 10 min at 4 °C using a Sorvall
LYNX 4000 centrifuge (Thermo Fisher Scientific, Waltham, MA), and
the cell pellets were stored at −20 °C until used.
5
Alanine Scanning Mutagenesis of MC-FN-010
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For generation of alanine scanning MC-FN-010 derivatives mutations were either introduced via PCR or the whole coding sequence was assembled via direct synthesis of GeneArt™ Strings™ fragments (Thermo Fisher Scientific). Respective DNA fragments were cloned into pET-32-LibEx expression vector using unique BamHI and KpnI restriction sites and introduced into E. coli SHuffle® T7 Express competent cells (New England BioLabs). All mutations were verified by DNA sequencing. The alanine scanning mutagenesis variants were expressed in 24-well format using 5 mL of selective autoinduction medium. Production and Trx-cystine-knot miniprotein purification were performed as described above for the 96-well format, but included a further purification step of the supernatant using HisPur™ Ni-NTA spin columns (Thermo Fisher Scientific). Binding ability and specificity of Trx-cystine-knot miniproteins to target and off-target protein was carried out with an antibody-based ELISA assay as described above.
6
Overexpression of Thermoplasma acidophilum Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
E. coli SHuffle
T7 Express competent cells and restriction enzymes were obtained from
the New England Biolabs (Ipswich, MA). Thermoplasma
acidophilum genomic DNA and pTev5 were generous gifts
from Dr. Dennis G. Searcy of University of Massachusetts Amherst and
Dr. Jorge Escalante-Semerena of the University of Georgia, respectively.
All chemicals were purchased from either Sigma-Aldrich (St. Louis,
MO) or Fisher Scientific (Waltham, MA).
T7 Express competent cells and restriction enzymes were obtained from
the New England Biolabs (Ipswich, MA). Thermoplasma
acidophilum genomic DNA and pTev5 were generous gifts
from Dr. Dennis G. Searcy of University of Massachusetts Amherst and
Dr. Jorge Escalante-Semerena of the University of Georgia, respectively.
All chemicals were purchased from either Sigma-Aldrich (St. Louis,
MO) or Fisher Scientific (Waltham, MA).
7
Purification and Characterization of rRsBBI1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pET23a-RsBBI1 plasmid was transformed into E. coli SHuffle T7 express competent cells (NEB, UK) by heat shock method. These host cells have a chromosomal copy of constitutively-expressed disulphide bond isomerase (DsbC), a chaperone assisting in proper folding of the cytoplasmic proteins (de Marco, 2009) . Single colony transformants were cultured in LB-Amp broth and incubated at 37 C. The overnight grown culture was inoculated into 1 L of LB-Amp broth on the following day and incubated at 37 C until the culture reaches ~1.0 OD 600 units. Later, the culture was induced using 0.4 mM isopropyl-b-D-thiogalactoside (IPTG) at 30 C for 8 h to express the rRsBBI1. The cell pellet obtained was suspended in 50 mM Tris-HCl (pH 8.0) containing 500 mM NaCl and sonicated. The lysate was heated at 80 C for 30 min and chilled on ice. The rRsBBI1 was purified from the supernatant collected after centrifugation at 10,000 g using trypsin coupled CNBr-Sepharose column in fast protein liquid chromatography (FPLC) AKTAprime plus (1 mL Flow rate at 25 C and 1 Bar pressure). The rRsBBI1 eluted with 0.01 N HCl was neutralized with 50 mM Tris-HCl (pH 8.0), concentrated and stored at À20 C until further use. The purification profile was represented in 15% SDS-PAGE as per Laemmli (1970) and in-gel trypsin inhibitor activity of rRsBBI1 was visualized as described by Felicioli et al. (1997) .
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
Revolutionizing how scientists
search and build protocols!