Journal: Applied Microbiology and Biotechnology

Article Title: A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

doi: 10.1007/s00253-019-10145-1

Figure Lengend Snippet: Controlled induction of sfGFP expression. a Fluorescence quantification of sfGFP production with increasing amounts of rhamnose in shaker flasks at different time points: pre-induction (white), 5 h (gray), or 7 h (black) after induction. The E. coli pSAR-2 strain without sfGFP insert is indicated as “Empty”. Error bars indicate standard deviations of two biological replicates. b Flow cytometry analysis (FACS) data of E. coli BL21 cells with pSAR-2 empty and pSAR-2::sfGFP vector expressing sfGFP, 4 h after induction with different amounts of rhamnose in shaker flasks. Left plots showing cell granularity by forward scatter (FSC-A, x -axis) and side scatter (SSC-A, y -axis) per cell. Right histograms showing the number of cells per mean GFP fluorescence intensity (au). c Scalability of sfGFP expression in different culturing systems induced at OD 600nm 0.6–0.8 with 10 mM rhamnose

Article Snippet: Bacterial strains, plasmids, and growth conditions Escherichia coli JM109 (Sambrook et al. ) was used for routine cloning procedures and E. coli BL21 (Novagen/Merck, Darmstadt, Germany ) for expression of the recombinant proteins.

Techniques: Expressing, Fluorescence, Flow Cytometry, Cytometry, FACS, Plasmid Preparation

Journal: Applied Microbiology and Biotechnology

Article Title: A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

doi: 10.1007/s00253-019-10145-1

Figure Lengend Snippet: SDS-PAGE and Western blot analysis of antibody affinity chromatography purification of PGT135 scFv. Analysis of feed, flow-through (FT), wash, and elution fractions 8–11 (E8–E11) of an affinity chromatography run using Capto-L for purification of PGT135 scFv antibody fragment from the soluble protein fraction of E. coli BL21 cells expressing PGT135 scFv from pSAR-2 vector. a Representative SDS-PAGE gel showing total protein in different analyzed fractions and PGT135 scFv antibody with an expected band size of 29 kDa. Elution fractions show highly enriched PGT135 scFv after purification. b Representative western blot with HRP–Protein L binding to PGT135 scFv in different analyzed fractions. In the first lane, 2.8 μg of commercially available His-tagged PGT135 scFv (scFv-His 6 xHis) is included for semi-quantification. PGT135 scFv is clearly visible and semi quantified in feed and elution fractions

Article Snippet: Bacterial strains, plasmids, and growth conditions Escherichia coli JM109 (Sambrook et al. ) was used for routine cloning procedures and E. coli BL21 (Novagen/Merck, Darmstadt, Germany ) for expression of the recombinant proteins.

Techniques: SDS Page, Western Blot, Affinity Chromatography, Purification, Flow Cytometry, Expressing, Plasmid Preparation, Binding Assay

Journal: Applied Microbiology and Biotechnology

Article Title: A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

doi: 10.1007/s00253-019-10145-1

Figure Lengend Snippet: Expression of antibody fragment PGT135 scFv. PGT135 scFv is expressed by E. coli BL21 from the pSAR-2:scFv vector with different concentrations of rhamnose for induction, and at two different temperatures (30 °C and 25 °C) and harvest times (21 h and 48 h). a Representative western blot with Protein L–HRP conjugate binding to PGT135 scFv antibody fragments in the soluble protein fraction. Numbers 3, 10, and 15 indicate the amount of rhamnose added for induction in mM. E. coli BL21 pSAR-2 empty induced with 10 mM of rhamnose is included as a negative control. Two different known concentrations of the commercially available His 6 -tagged antibody PGT135 scFv-6xHis are included for quantification. b Graph showing semi-quantitative data for PGT135 scFv production in shaker flasks as determined by western blot based on two biological replicates. Black bars show PGT135 scFv in total cell fraction and gray bars in soluble protein fraction. Error bars indicate standard deviation of two biological duplicates

Article Snippet: Bacterial strains, plasmids, and growth conditions Escherichia coli JM109 (Sambrook et al. ) was used for routine cloning procedures and E. coli BL21 (Novagen/Merck, Darmstadt, Germany ) for expression of the recombinant proteins.

Techniques: Expressing, Plasmid Preparation, Western Blot, Binding Assay, Negative Control, Standard Deviation