Cloning, expression, and protein purification were carried out as described earlier (18, 20). Briefly, DNA sequences encoding bacterial family three CBMs (CBM3) from Ruminiclostridium thermocellum (Protein Data Bank accession number 1NBC) (21), DNA sequence encoding a 499-amino acid stretch of the A. diadematus dragline spidroin (ADF3), and a 12-time repeat of residues 325 to 368 in ADF3 (eADF3) (11, 35, 36) were synthesized and codon-optimized by GeneArt Gene Synthesis (Thermo Fisher Scientific) for expression in Escherichia coli. Standard methods of molecular cloning were used (37). Resulting constructs were named CBM-eADF3-CBM, CBM-ADF3-CBM, CBM-eADF3, CBM-CBM, and CBM. XL1B [New England BioLabs 5-alpha F′Iq Competent E. coli (F′ proA+B+lacIq(lacZ)M15 zzf::Tn10 (TetR)/fhu A2(argF-lacZ)U169 phoA glnV44 Φ80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17)] and BL21 [(F-ompT hsdSB (rB-mB-) gal dcm (DE3), Thermo Fisher Scientific] were used as cloning and expression strains, correspondingly. Either EnPresso B medium (BioSilta, Oulu, Finland) or MagicMedia (Thermo Fisher Scientific) was used as expression media. After 15 to 24 hours of induction, cells were harvested by centrifugation (16,000g, 15 min, 4°C), washed, and lysed. Correspondingly, the eADF3 sequence was synthesized and codon-optimized by GeneArt Gene Synthesis for expression in Pichia pastoris. The codon-optimized sequence was inserted in pPICZα (Invitrogen) expression vectors and then transferred into TOP10 E. coli cells (Thermo Fisher Scientific) and selected on Zeocin low-salt LB plates. After initial screening, assembled plasmids were linearized and transformed into the X33 P. pastoris strain selected for on Zeocin plates and then cultured overnight in buffered glycerol-complex expression medium. The cells were harvested by centrifugation (1000g, 5 min) after the density reached an OD600 (optical density value at a wavelength of 600 nm) value of 3 to 6. The protein production was initiated by diluting the cells to OD600 of 1.0 in 250 ml of buffered methanol-complex medium in 2-liter flasks, followed by the addition of protease inhibitors chymostatin and pepstatin A to final concentrations of 1 μM each. Methanol was added daily to uphold a concentration of 2% (v/v), which was necessary for induction of protein expression. All proteins were purified either with HisTrap FF crude columns (GE Healthcare Life Sciences) connected to an ÄKTA pure liquid chromatography system or by heat fractionation at 70° to 75°C for 30 min, followed by centrifugation at 16,000g for 80 min at 4°C. Fractions containing the protein of interest were desalted using Econo-Pac 10DG columns. Samples were then frozen in liquid nitrogen and stored at −80°C until use.

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