Shuffle t7 express e coli

The MUC16 sequence cited by O’Brien et al. [30 ] was sourced from NCBI (GenBank AF414442.2) and used to derive the nucleotide sequences of the nine tandem repeats used in this study (sequences found in Supplementary Information Table S1). The sequences were synthesized and cloned into pET14b vectors with XhoI and BamHI sites (plasmids purchased from GenScript, Piscataway, NJ, USA) to express individual tandem repeat proteins with N’ 6xHis-tagging. Plasmids were transformed into Shuffle T7 Express E. coli (New England Biolabs, Beverly, MA, USA), and clones were grown to exponential phase in Luria-Bertani (LB) broth (Thermo, Waltham, MA, USA) with 100 μg/mL of ampicillin at 30 °C. Protein expression was induced by adding 400 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h. Cells in phosphate-buffered saline (PBS) buffer with cOmpletet^TM protease inhibitor (Thermo) were harvested and lysed via freeze–thaw cycles. Ni²⁺-NTA beads (Thermo) were used to purify the 6xHis-tagged repeat proteins. Protein identities were verified via 6xHis antibody Western blots and mass spectrometry as described in our previous work [24 (link)] and as demonstrated in Supplementary Information Table S1.

Impranil DLN W 50 agar plates were used for the screening of the IsPETaseTM mutant library. The lysogeny broth (LB) agar plates contained 0.5 mM isopropyl‐β‐d‐thiogalactopyranoside (IPTG), 100 μg mL⁻¹ ampicillin, and 0.5% Impranil DLN W 50, diluted from a 40% suspension. The mutant library was used to transform chemically competent E. coli SHuffle T7 Express (New England Biolabs GmbH, Frankfurt am Main, Germany) cells which were then spread onto LB agar plates containing 100 μg mL⁻¹ ampicillin. After the cells were incubated overnight at 30°C, colonies were picked and transferred to another LB‐ampicillin agar plate (storage plate) and in parallel to the Impranil agar plate (assay plate). The storage and assay plates were first incubated overnight at 30°C, the assay plates were subsequently incubated at 60°C for 24 h. Degradation of Impranil leads to formation of clear zones around colonies expressing active IsPETase variants. An increased size of the haloes formed, relative to the TM as control, was used as a preliminary indication of increased thermostability. Selected colonies were picked from the storage plate and used to inoculate an overnight culture for plasmid isolation using the innuPREP Plasmid Mini Kit (Analytik Jena GmbH, Jena, Germany). Increased thermostability was then experimentally verified by protein expression, purification and nanoDSF, as described below.

All chemical reagents were molecular biology grade. Phage display scFv library from nonimmunized human, Yamo I was constructed in our laboratory using B-lymphocytes from 140 healthy individuals in the Northeastern Thailand (12 (link)). E. coli TG1 was obtained from MRC, Cambridge, and was used for cloning and amplification of phages. E. coli SHuffle T7 Express (New England Biolabs [NEB], USA) was used for protein expression. The anti-M13/horseradish peroxidase (HRP) and His-probe-HRP were purchased from Amersham-Pharmacia Biotech (Uppsala). Anti-His-Dylight 488 secondary antibody and Calcofluor white stain were purchased from Abcam and Sigma, respectively. Bradyrhizobium strains SUTN9-2, DOA9 and other Bradyrhizobium strains were obtained from School of Biotechnology, Suranaree University of Technology.