CpoI (RsrII) restriction enzyme

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	The expression vector of P. pastoris pHBM905A with 5′AOX1 gene promoter was digested by CpoI and NotI restriction enzymes.

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The expression vector of P. pastoris pHBM905A with 5′AOX1 gene promoter was digested by CpoI and NotI restriction enzymes.

Publication protocol

"In order to further increase the expression level of xylanase in P. pastoris, the gene sequence of xynHBN188A was optimized based on the codon usage bias of P. pastoris. The codon-optimized gene was designed by DNAworks (http://helixweb.nih.gov/dnaworks/) and synthesized by Genscript (China). Primers HBsF and HBsR (Table 1) were used to amplify xynHBN188As gene and HB3 and HB4 primers (Table 1) for xynHBN188A. The partial CpoI and NotI (TaKaRa) are underlined. The PCR products were treated with T4 DNA Polymerase and 1mM dTTP for 20min at 12 °C for overhangs. The expression vector of P. pastoris pHBM905A with 5′AOX1 gene promoter was digested by CpoI and NotI restriction enzymes35. The treated PCR products were ligated with pHBM905A downstream of the MFα secretion signal sequence. The ligation products were transformed into E. coli DH5α. The transformants were screened on LB plates (100μg/mL ampicillin), and further confirmed by colony PCR and DNA sequencing.
The recombinant plasmids (Figure S1) were linearized using SalI and transformed into P. pastoris GS115 by electroporation (10000 V/cm, 4 ms, Bio-Rad MicroPulser Electroporator, USA). Transformants were selected on histidine-deficient MD plates and incubated at 30 °C for 3 days. Positive transformants were identified on an upside down BMMY plate containing 0.5% Remazol Brilliant Blue (RBB)-xylan with methanol dropping uniformly on the petri dish cover at 28 °C. Three colonies with the minimum halo were selected for shake flask fermentation as described in previous report with minor modification31. Cells were cultured in 25ml of BMGY medium for 48h and harvested. They were transferred into 25ml of BMMY medium to induce xylanase expression and measured at regular intervals. Three replicates were performed for each transformant to test the activity."

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