Journal: MethodsX

Article Title: Optimization of production of recombinant gamma-tubulin in bacteria

doi: 10.1016/j.mex.2021.101517

Figure Lengend Snippet: Optimizing the conditions for expressing recombinant TUBG1: (A) a scheme for the optimization of culture conditions for the expression of TUBG1 using ampicillin (Amp) as a selection marker in transformed E . coli. In short, a selected E. coli colony carrying a vector containing the TUBG1 and Amp genes is cultured overnight (ON) in Luria–Bertani liquid supplemented with ampicillin. Then, the bacteria are diluted and cultured until the optical density measured at a wavelength of 600 nm (OD 600 ) reaches 0.6 to 0.8. The expression of recombinant TUBG1 is induced with isopropyl-1-thio-β-D-galactopyranoside (IPTG) during the indicated times and conditions. GST-TUBG1 (B) and TUBG1-His 6 (C) were induced in E. coli DH5α containing the pGEX2T- TUBG1 vector with 0.2 mM IPTG (B) and in E. coli BL21 carrying the pET- TUBG1 -His 6 with 1 mM IPTG (C), respectively. Thereafter, the bacteria were incubated under the following conditions: 1 h at 37 °C (1 h, 37 °C), overnight incubation at room temperature (ON, RT), and 1 h at 37 °C followed by overnight incubation at room temperature (1 h, 37 °C ON, RT). (B and C) The expression of recombinant TUBG1 was analyzed by Western blot (WB) using an anti-TUBG1 antibody. The highest yield of GST-TUBG1 was obtained upon stimulation with 0.2 mM IPTG for 1 h at 37 °C followed by overnight incubation at room temperature (B), whereas the highest yield of TUBG1-His 6 was reached after stimulation with 1.0 mM IPTG for 1 h at 37 °C (C). (B and C) The graphs illustrate densitometric analysis of the TUBG1 content in the Western blots presented (mean ± SD; N = 3, ** P < 0.01, * P < 0.05).

Article Snippet: Purification of GST- and His 6 -tagged human TUBG1 from bacteria lysates Step 1: Optimization of the culture conditions with small-scale expression cultures Materials ▪ pGEX2T- TUBG1 (Addgene Plasmid # 171967) ▪ pET21- TUBG1 (Addgene Plasmid # 101825) ▪ E. coli DH5α (gives high protein yield from pGEX2T- TUBG1 plasmid; Thermo Fisher Scientific, cat. no. 18265017) ▪ E. coli BL21([DE3] optimized for protein expression from the T7 promoter included in a pET21 plasmid; Thermo Fisher Scientific, cat. no. C600003) ▪ Sterile Luria–Bertani liquid (LB) medium (Merck, cat. no. L2542) ▪ Sterile LB agar plates (Sigma-Aldrich, cat. no. L5667) ▪ Ampicillin (Thermo Fisher Scientific, cat. no. 11593027) dissolved in sterile, deionized distilled water ▪ Sterile 14 ml polypropylene round-bottom tubes (Fisher scientific; Falcon, cat. no. 10384641) ▪ Isopropyl-β-D- thiogalactoside (IPTG; Sigma-Aldrich, cat. no I6758) dissolved in sterile, deionized distilled water ▪ Phenylmethylsulphonyl fluoride (PMSF; Sigma-Aldrich, cat. no 10837091001) dissolved according to the manufacturer's instructions ▪ Anti-TUBG (1:1000; Sigma-Aldrich, cat. no T3320) ▪ Sterile phosphate-buffered saline (PBS; Sigma-Aldrich, cat. no. P4417) ▪ 1.5 ml microcentrifuge tubes (Sigma-Aldrich, cat. no. Z336777) ▪ Shaking incubator for culture growth (Merck, cat. no. CLS6791) ▪ Soniprep 150 Plus with exponential probe (240V; MSE, cat. no. MSS150.CX4.5) ▪ 5X loading buffer: 625 mM TRIS pH 6.5 (Sigma-Aldrich, cat. no. 10812846001), 10% (w/v) sodium dodecyl sulfate (SDS; Sigma-Aldrich, cat. no. GE17-1313-01), 25% (v/v) glycerol (Sigma-Aldrich, cat. no. G5516), 0.005% (w/v) bromophenol blue (Sigma-Aldrich, cat. no. GE17-1329-01), 250 mM dithiothreitol (Sigma-Aldrich, cat. no. D0632), and deionized distilled water Note that the preceding list includes only the necessary bacteria strains and non-standard laboratory equipment.

Techniques: Expressing, Recombinant, Selection, Marker, Transformation Assay, Plasmid Preparation, Cell Culture, Bacteria, Incubation, Western Blot

Journal: MethodsX

Article Title: Optimization of production of recombinant gamma-tubulin in bacteria

doi: 10.1016/j.mex.2021.101517

Figure Lengend Snippet: Purification of GST-TUBG1 and removal of the GST tag: a scheme of the production and affinity purification of GST-TUBG from E. coli DH5α carrying the pGEX2T- TUBG1 vector and using ampicillin (Amp) as a selection marker. In short, the E. coli colony from a pre-selected colony is cultured overnight (ON) in Luria–Bertani liquid supplemented with ampicillin. Then, the bacteria are diluted and cultured until the optical density measured at a wavelength of 600 nm (OD 600 ) reaches 0.6 to 0.8. The expression of recombinant TUBG1 is induced with 0.2 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 1 h at 37 °C followed by overnight incubation at room temperature. The bacteria are harvested and lysed by sonication. After preclearing the lysates by centrifugation (centrif.), the recombinant GST-TUBG1 in the resulting bacterial lysates (lysates) is affinity purified with Glutathione Sepharose 4B beads. The GST tag is thereafter cleaved with thrombin after removal of PMSF (Dirty beads) or after extensive washing (Washed beads). The expression of recombinant TUBG1 and the efficient removal of GST were analyzed by SDS-page stained with Gelcode Blue and by Western blotting (WB) using an anti-TUBG1 antibody and anti-GST antibodies. The color of the arrows shows the sequence of events in the scheme (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Article Snippet: Purification of GST- and His 6 -tagged human TUBG1 from bacteria lysates Step 1: Optimization of the culture conditions with small-scale expression cultures Materials ▪ pGEX2T- TUBG1 (Addgene Plasmid # 171967) ▪ pET21- TUBG1 (Addgene Plasmid # 101825) ▪ E. coli DH5α (gives high protein yield from pGEX2T- TUBG1 plasmid; Thermo Fisher Scientific, cat. no. 18265017) ▪ E. coli BL21([DE3] optimized for protein expression from the T7 promoter included in a pET21 plasmid; Thermo Fisher Scientific, cat. no. C600003) ▪ Sterile Luria–Bertani liquid (LB) medium (Merck, cat. no. L2542) ▪ Sterile LB agar plates (Sigma-Aldrich, cat. no. L5667) ▪ Ampicillin (Thermo Fisher Scientific, cat. no. 11593027) dissolved in sterile, deionized distilled water ▪ Sterile 14 ml polypropylene round-bottom tubes (Fisher scientific; Falcon, cat. no. 10384641) ▪ Isopropyl-β-D- thiogalactoside (IPTG; Sigma-Aldrich, cat. no I6758) dissolved in sterile, deionized distilled water ▪ Phenylmethylsulphonyl fluoride (PMSF; Sigma-Aldrich, cat. no 10837091001) dissolved according to the manufacturer's instructions ▪ Anti-TUBG (1:1000; Sigma-Aldrich, cat. no T3320) ▪ Sterile phosphate-buffered saline (PBS; Sigma-Aldrich, cat. no. P4417) ▪ 1.5 ml microcentrifuge tubes (Sigma-Aldrich, cat. no. Z336777) ▪ Shaking incubator for culture growth (Merck, cat. no. CLS6791) ▪ Soniprep 150 Plus with exponential probe (240V; MSE, cat. no. MSS150.CX4.5) ▪ 5X loading buffer: 625 mM TRIS pH 6.5 (Sigma-Aldrich, cat. no. 10812846001), 10% (w/v) sodium dodecyl sulfate (SDS; Sigma-Aldrich, cat. no. GE17-1313-01), 25% (v/v) glycerol (Sigma-Aldrich, cat. no. G5516), 0.005% (w/v) bromophenol blue (Sigma-Aldrich, cat. no. GE17-1329-01), 250 mM dithiothreitol (Sigma-Aldrich, cat. no. D0632), and deionized distilled water Note that the preceding list includes only the necessary bacteria strains and non-standard laboratory equipment.

Techniques: Purification, Affinity Purification, Plasmid Preparation, Selection, Marker, Cell Culture, Bacteria, Expressing, Recombinant, Incubation, Sonication, Centrifugation, SDS Page, Staining, Western Blot, Sequencing