vitro protein synthesis ivps kit  (New England Biolabs)

Journal: bioRxiv

Article Title: Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters

doi: 10.1101/2020.04.04.025353

Figure Lengend Snippet: Synthesis of BpsA with the PURE cell-free system. (A) Expression control by Western blotting with anti-Strep antibodies performed in three independent reaction solutions (#1-3). BpsA was applied as holo -protein, produced by IVPS with simultaneous phosphopantetheinylation. Self-cast 9 % Tris-Tricine gel. Strep-tagged BpsA has a molecular weight of 142.7 kDa. For the uncropped blot, see Figure S2A. (B) SEC profiles and Western Blot detection of elution fractions. (top) Recombinantly produced BpsA and (bottom) IVPS reaction solution including phosphopantetheinylation. (C) Quantification of protein production yields and phosphopantetheinylation efficiency. BpsA was first produced by IVPS and then phosphopantetheinylated with Sfp and CoA-647 (purchased from NEB). Samples from three independent reactions (#1-3) were applied in repetition (a b). For calibration, recombinantly produced BpsA, diluted in the PURExpress reaction solution, was loaded in amounts of 1.25, 0.63, 0.31 and 0.16 pmol. 9 % Tris-Tricine gel as in panel A. For the uncropped gels, see Figure S2B. Overall, three times three reactions, each applied in duplicate (18 bands), were used for quantification of BpsA production and phosphopantetheinylation for the parallel and the sequential protocol, respectively (Figure S3 A-C).

Article Snippet: In evaluating the PURE system for the cell-free synthesis of natural compounds from genomic DNA, we worked with the commercially available E. coli -based PURExpress In Vitro Protein Synthesis (IVPS) Kit as a “reaction solution” for gene expression and product formation (New England Biolabs, USA) .

Techniques: Expressing, Western Blot, Produced, Molecular Weight