Journal: Molecules

Article Title: Selecting Nanobodies Specific for the Epidermal Growth Factor from a Synthetic Nanobody Library

doi: 10.3390/molecules28104043

Figure Lengend Snippet: Production of (rEGF) in E. coli . ( a ) Amino-acid sequence of rEGF with C-terminal tags: cyan—EGF, yellow—SV5 tag, orange—6xHis tag. ( b ) Scheme of the pET22b-rEGF expression vector. ( c ) SDS-PAGE and Western blot of the production of rEGF in BL21 (DE3). The rEGF was identified using an anti-His tag monoclonal antibody conjugated to HRP. M: molecular-weight marker, Trident Prestained Protein Ladder GTX50875 (GeneTex, USA), 1: untransformed BL21, 2: BL21 transformed with pET22b-rEGF. ( d ) SDS-PAGE of the rEGF-soluble fraction obtained by cell lysis using several freezing/thawing rounds. M: molecular-weight marker, 1: total fraction of BL21 transformed with pET22b-hEGF, 2: soluble fraction of BL21 transformed with pET22b-hEGF. ( e ) SDS-PAGE of the rEGF-purification process. Left panel: first purification stage by IMAC. M: molecular-weight marker, 1: initial sample, 2: unbound proteins, 3: wash (40 mM imidazole), 4: elution (250 mM imidazole). Right panel: second purification stage with IEC. M: molecular-weight marker, 1: initial sample from IMAC elutions, 2, 3, and 4: unbound proteins, 5: elution from the anionic exchanger Bio-Scale Mini Macro-Prep High Q Cartridge (BioRad, Hercules, CA, USA). ( f ) In vitro evaluation of rEGF biological activity in A431 cells. Two FBS concentrations were used: (0.5%—blue bars; and 5%—red bars). Measurements were performed in triplicate. Stars indicate statistically significant differences between experimental conditions (* p < 0.05, ** p < 0.005).

Article Snippet: The gene coding the EGF was synthesized and cloned into the pET22b-expression vector (pET22b-EGF) by GenScript (Piscataway, NJ, USA).

Techniques: Sequencing, Expressing, Plasmid Preparation, SDS Page, Western Blot, Molecular Weight, Marker, Transformation Assay, Lysis, Purification, In Vitro, Activity Assay

Journal: Molecules

Article Title: Selecting Nanobodies Specific for the Epidermal Growth Factor from a Synthetic Nanobody Library

doi: 10.3390/molecules28104043

Figure Lengend Snippet: Production and purification of anti-EGF nanobodies. ( a ) SDS-PAGE and Western blot of nanobody production in BL21 (DE3). M: molecular-weight marker, PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, USA), 1: untransformed BL21; BL21 transformed with (2) pET22b-NbA8-1, (3) pET22b-NbA11-2, and (4) pET22b-NbD6-4. The nanobodies were identified using an anti-His tag monoclonal antibody conjugated to HRP. ( b ) SDS-PAGE of the nanobody-purification process. Left panel: first purification stage by IEC. M: molecular-weight marker, 1: initial sample, 2: unbound proteins, 3: elution from the anionic exchanger, Bio-Scale Mini Macro-Prep High Q Cartridge (BioRad, USA), 4: elution from the cationic exchanger CM Sepharose Fast Flow (GE Healthcare, USA). Right panel: second purification stage by IMAC. M: molecular-weight marker, 1: initial sample from IEC, 2: unbound proteins, 3: wash (40 mM imidazole), 4: elution (250 mM Imidazole). Arrow heads indicate the protein bands corresponding to nanobodies A8-1, A11-2, and D6-4.

Article Snippet: The gene coding the EGF was synthesized and cloned into the pET22b-expression vector (pET22b-EGF) by GenScript (Piscataway, NJ, USA).

Techniques: Purification, SDS Page, Western Blot, Molecular Weight, Marker, Transformation Assay

Journal: Microbial Cell Factories

Article Title: Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins

doi: 10.1186/s12934-015-0241-5

Figure Lengend Snippet: SDS-PAGE gels of ApoA-1 and Hsp70. The proteins were expressed in endotoxin-free derivatives of E. coli strains BW30270 (A) and BL21 (DE3) (B) , respectively, and minimally purified using IMAC on HisTrap HP (1 ml) columns. The recombinant ApoA-1 and Hsp70 samples (6 μg each) were resolved under denaturing conditions using 12% and 10% polyacrylamide gels, respectively. Molecular mass protein markers (kDa) were run in lanes M. Arrows indicate the positions of ApoA-1 and Hsp70.

Article Snippet: The codon-optimized gene for human Hsp70 was synthesized by GenScript, Piscataway, NJ, digested with Nde I and Hind III, and ligated into the Nde I/ Hind III sites of pET-22b to yield plasmid pHsp70His.

Techniques: SDS Page, Purification, Recombinant

Journal: Microbial Cell Factories

Article Title: Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins

doi: 10.1186/s12934-015-0241-5

Figure Lengend Snippet: Reactivity of ApoA-1 and Hsp70 in the Limulus amebocyte lysate (LAL) assay. ApoA-1 was produced in E. coli strain BW30270/pApo404 and its endotoxin-free derivatives KPM318/pApo404 and KPM335/pApo404, whereas Hsp70 was obtained from KPM404/pHsp70His and its parental strain BL21 (DE3)/pHsp70His. The proteins were minimally purified by IMAC and assayed with the LAL test. The measurements represent the means and standard deviations from three individual experiments.

Article Snippet: The codon-optimized gene for human Hsp70 was synthesized by GenScript, Piscataway, NJ, digested with Nde I and Hind III, and ligated into the Nde I/ Hind III sites of pET-22b to yield plasmid pHsp70His.

Techniques: LAL Assay, Produced, Purification

Journal: Microbial Cell Factories

Article Title: Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins

doi: 10.1186/s12934-015-0241-5

Figure Lengend Snippet: Stimulation of hTLR4/MD-2 by ApoA-1 and Hsp70 produced in endotoxin-free E. coli strains. The proteins were minimally purified by IMAC and assayed with HEK-Blue hTLR4 cells for their ability to activate NF-κB-dependent SEAP expression (A and C) . HEK-Blue Null2 cells served as a control (B and D) . Relative NF-κB induction was measured following stimulation of HEK-Blue hTLR4 and Null2 cells with tenfold serial dilutions of ApoA-1 (A and B) and Hsp70 (C and D) samples obtained by heterologous expression in BW30270/pApo404, KPM318/pApo404 and KPM335/pApo404, and BL21 (DE3)/pHsp70His and KPM404/pHsp70His, respectively. The values represent the means and standard deviations from three individual experiments. In all experiments, the ApoA-1 and Hsp70 samples did not activate NF-κB-dependent SEAP expression in HEK-Blue Null2 cells, suggesting that NF-κB-dependent SEAP expression was due to specific activation of the hTLR4/MD-2 signaling pathway in HEK-Blue hTLR4 cells.

Article Snippet: The codon-optimized gene for human Hsp70 was synthesized by GenScript, Piscataway, NJ, digested with Nde I and Hind III, and ligated into the Nde I/ Hind III sites of pET-22b to yield plasmid pHsp70His.

Techniques: Produced, Purification, Expressing, Control, Activation Assay

Journal: Biotechnology for Biofuels

Article Title: Iron incorporation both intra- and extra-cellularly improves the yield and saccharification of switchgrass ( Panicum virgatum L.) biomass

doi: 10.1186/s13068-021-01891-4

Figure Lengend Snippet: Protein purification of iron-binding fusion proteins and iron-binding assay. a The SDS-PAGE analysis of SP GRP -CBM11-IBP and SP GRP - CBM11-4xIBP proteins purified from recombinant E. coli cells. b Comparison of the iron-binding ability of iron-binding fusion proteins at pH 5.5 on a PVDF membrane. c Comparison of the iron-binding ability of iron-binding fusion proteins at pH 7.0 using light absorbance at 510 nm

Article Snippet: Synthesized DNA fragments (SP GRP -CBM11-IBP-His 6 and SP GRP -CBM11-4xIBP-His 6 ) were cloned into a pET-22b (+) vector (Genscript, Piscataway, NJ) and transformed into Escherichia coli BL21 for protein expression.

Techniques: Protein Purification, Binding Assay, SDS Page, Purification, Recombinant

Journal: Journal of Bacteriology

Article Title: The Essential β-Barrel Assembly Machinery Complex Components BamD and BamA Are Required for Autotransporter Biogenesis ^▿

doi: 10.1128/JB.00192-11

Figure Lengend Snippet: BamB, BamC, and BamE are not required for AT secretion. (A) Construction of bamB, bamC, and bamE null mutations in E. coli HB101 and E. coli CFT073. Whole-cell lysate of each mutant was prepared by spinning down 1 ml of an overnight culture and resuspending it in Laemmli buffer. Samples were separated by SDS-PAGE and Western immunoblotted with antibodies raised to BamB, BamC, and BamE, confirming the absence of the relevant protein from each mutant. (B) Western immunoblotting was performed on the BamB, BamC, and BamE null mutant strains expressing Pet and Ag43. The Pet passenger domain was prepared from supernatant fractions after filter sterilization and precipitation with a final volume of 10% trichloroacetic acid. No difference in the accumulation of Pet in culture supernatant was noted. The Pet β-barrel was detected by Western immunoblotting of outer membrane fractions with anti-Pet β-domain antibodies after heating to either 100°C or 25°C; no differences in β-domain levels or heat modifiability were detected. For analysis of Ag43 biogenesis, the Ag43 passenger domain was released from the cell surface using the standard heat release assay (60°C for 2 min) and detected by Western immunoblotting with Ag43-specific antiserum. No differences in the levels of Ag43 were detected. These data indicate that AT secretion is not significantly influenced by BamB, -C, or -E. WT, wild type.

Article Snippet: Anti-BamE antibodies were previously described ( 21 , 23 ); to raise antibodies to BamB and BamC, constructs encoding these proteins but lacking their acylation site were synthesized de novo and cloned into the vector pET22b + (Genscript).

Techniques: Mutagenesis, SDS Page, Western Blot, Expressing, Membrane, Release Assay

Journal: Journal of Bacteriology

Article Title: The Essential β-Barrel Assembly Machinery Complex Components BamD and BamA Are Required for Autotransporter Biogenesis ^▿

doi: 10.1128/JB.00192-11

Figure Lengend Snippet: BamB, BamC, and BamE are not required for folding of the passenger domains. If the AT passenger domains do not fold correctly, they will not be able to perform their extracellular functions. To determine if BamB, BamC, or BamE influences the folding of the passenger domain, the functional activity of the Pet and Ag43 ATs was characterized in wild-type (WT) E. coli HB101 and isogenic derivatives. (A) The enzymatic activity of Pet was calculated using an azocasein assay. Protease activity was expressed in arbitrary units. There was no difference in the functional activity of Pet harvested from the mutant strains and that of Pet harvested from the wild-type organism, indicating that Pet produced from the mutants is not aberrantly folded. (B) The ability of Ag43 to mediate cell-cell aggregation was quantified in wild-type E. coli HB101 and isogenic derivatives. No difference in the rate of sedimentation between the mutants and the parent strain was observed. In contrast, the E. coli HB101 strain lacking the gene encoding Ag43 (Δflu) did not aggregate at all. These data indicate that Ag43 is not aberrantly folded.

Article Snippet: Anti-BamE antibodies were previously described ( 21 , 23 ); to raise antibodies to BamB and BamC, constructs encoding these proteins but lacking their acylation site were synthesized de novo and cloned into the vector pET22b + (Genscript).

Techniques: Functional Assay, Activity Assay, Azocasein Assay, Mutagenesis, Produced, Sedimentation

Journal: Scientific Reports

Article Title: Myristoyl group-aided protein import into the mitochondrial intermembrane space

doi: 10.1038/s41598-018-38016-1

Figure Lengend Snippet: Mic19 import requires the CX 10 C motif and Tom20. ( A ) Amino-acid sequence alignment of the N-myristoylation domain and CHCH domain of Mic19 from Homo sapiens , Mus musculus , Danio rerio , and Saccharomyces cerevisiae . ( B ) Radiolabeled wild-type (WT) Mic19 and its C146S/C157S mutant were incubated with mitochondria for the indicated times at 25 °C. After PK treatment, mitochondria were subjected to SDS-PAGE and radioimaging. Bound proteins (−PK) and imported, protease-protected proteins (+PK) were quantified, and the amounts of the radiolabeled proteins added to each reaction were set to 100%. ( C ) Radiolabeled wild-type (WT) Mic19 and its mutants (C146S, C157S and C146S/C157S) were incubated with mitochondria for the indicated times at 25 °C. Then the mitochondria were treated with 50 μg/ml PK and 50 mM IAA (2-iodoacetamide) for 20 min on ice and subjected to SDS-PAGE with 5 M urea in the absence (non-reducing) or presence (reducing) of 5% β-mercaptoethanol, and radioimaging. Arrowheads and asterisk indicate Mic19-Tim40 conjugates and nonspecific signals, respectively. In the lowermost panel, we incubated RI-labeled Mic19-C146S with mitochondria containing WT Tim40 or Tim40-FLAG for 20 min at 25 °C. After treatment with 50 mM IAA, the mitochondria were solubilized with 1% digitonin, and Tim40 conjugates were isolated by affinity purification for the FLAG tag. 5% of the applied sample (Load) and 100% of the eluted fraction (Elute) were analyzed by SDS-PAGE and radioimaging. Band shifts by the attached FLAG tag and pull-down by the anti-FLAG antibody confirmed that the 75 kD proteins (indicated with arrowheads in the uppermost panel), which were derived from WT Mic19 or Mic19C146S, contained Tim40 and thus represented Mic19-Tim40 conjugates. ( D ) The indicated proteins were analyzed by SDS-PAGE followed by immunoblotting for the wild-type strain (WT) and those with Tom20-AID*-9 × Myc and Tom20-3 × miniAID instead of Tom20 after cultivation in lactate medium (+0.05% glucose) at 30 °C. ( E ) The indicated radiolabeled proteins were incubated with the indicated mitochondria for the indicated times at 25 °C. After treatment with or without PK (50 μg/ml) for 20 min on ice, mitochondria were subjected to SDS-PAGE and radioimaging. Bound proteins (-PK) and imported, protease-protected proteins (+PK) were quantified, and the amounts of the radiolabeled proteins added to each reaction were set to 100%. Values are mean ± SEM ( n = 3). Full-length blot/gel images are presented in Supplementary Fig. .

Article Snippet: The E. coli strain BL21(DE3) transformed with the pET-22b plasmid (Merck Millipore) containing the gene for Tom20 sol -His, the cytosolic receptor domain of Tom20 from Rattus norvegicus (residues 51–145) with a hexa-histidine tag at the C-terminus, was cultured in LB medium containing 50 μg/ml ampicillin at 37 °C until OD 600 reached 0.5.

Techniques: Sequencing, Mutagenesis, Incubation, SDS Page, Labeling, Isolation, Affinity Purification, FLAG-tag, Derivative Assay, Western Blot

Journal: Scientific Reports

Article Title: Myristoyl group-aided protein import into the mitochondrial intermembrane space

doi: 10.1038/s41598-018-38016-1

Figure Lengend Snippet: DUF domain hampers import of TIM40/MIA pathway substrates, which is circumvented by N-myristoylation. ( A–E ) The indicated radiolabeled proteins were incubated with mitochondria for the indicated times at 25 °C. Bound and imported proteins were analyzed as in Fig. . ( F ) Mitochondria with Mic19WT-FLAG or Mic19G2A-FLAG were solubilized with 1% digitonin and incubated with 0.5μg of the purified cytosolic soluble domain of rat Tom20 (Tom20 sol ) bound to Ni-NTA resin or with Ni-NTA without bound Tom20 sol as a control. The bound proteins were eluted with 500 mM imidazole and subjected to SDS-PAGE and immunoblotting with the indicated antibodies. The amounts of Mic19WT-FLAG or Mic19G2A-FLAG added to each reaction were set to 100%. Values are mean ± SEM ( n = 3). Full-length gel images are presented in Supplementary Fig. .

Article Snippet: The E. coli strain BL21(DE3) transformed with the pET-22b plasmid (Merck Millipore) containing the gene for Tom20 sol -His, the cytosolic receptor domain of Tom20 from Rattus norvegicus (residues 51–145) with a hexa-histidine tag at the C-terminus, was cultured in LB medium containing 50 μg/ml ampicillin at 37 °C until OD 600 reached 0.5.

Techniques: Incubation, Purification, Control, SDS Page, Western Blot