Dmt chemically competent cells

Pichia pastoris GS115 was from Invitrogen (Shanghai, China). A site-directed mutagenesis kit and DMT chemically competent cells were from TransGen (Beijing, China). The R. chinensis lipr27RCL gene cloned in the pPIC9K vector was deposited in our laboratory. We obtained 4-nitrophenol palmitate (4-NPP) from Sigma. Mutagenic primers were synthesized by Shuoqing (Kunming, China). All other chemicals were commercially available analytical grade materials.

To analyze if the N-terminal Cys residue contributed to the dimerization of the sulfatase, the mutants of endoVB4SF-C27A and corresponding exoPB4SF-Q5C were produced using a Fast Mutagenesis System Kit (TransGen) as previously described by Prabhakar et al. (2005) (link) The plasmids were amplified using the primer pairs 5′-ATGGTGACTGCTGGCGCGTCGGCTAC-3′ (forward, endoVB4SF-C27A) and 5′-CGCGCCAGCAGTCACC ATGGTTGC-3′ (reverse, endoVB4SF-C27A), and 5′-TTCTTGA CGGGCAATTGCCCCGCTG-3′ (forward, exoPB4SF-Q5C) and 5′-GCAATTGCCCGTCAAGAATTCGGATCCG-3′ (reverse, exoPB4SF-Q5C). The plasmids of the mutants were amplified by transferring them to DMT Chemically Competent Cells (TransGen), and mutated clones were confirmed by DNA sequencing at Sangon Biotech Co., Ltd. (Shanghai, China). Next, the correct mutants were transferred into E. coli BL21 (DE3) cells. endoVB4SF-C27A and exoPB4SF-Q5C were expressed and purified as described above for the wild-type enzyme.
The purified endoVB4SF, exoPB4SF, endoVB4SF-C27A and exoPB4SF-Q5C proteins were detected at 280 nm through size-exclusion chromatography (Superdex^TM 200 10/300 GL, GE Healthcare) eluted with buffer (10 mM Tris-HCl and 100 mM NaCl, pH 8.0) with or without 5 mM DTT.

Site-directed mutations of sTAAR365 and mTAAR9 were introduced following the protocol of Quik Change site-directed mutagenesis kit (Agilent Technologies). In brief, PCR primers were designed by Agilent QuikChange Primer Design (Tables S3 and S4) and PCR reactions were performed using PfuUltra High-Fidelity DNA Polymerase with wild-type sTAAR365 and mTAAR9 plasmids as templates. The methylated parental strands were selectively digested with 1 μl DpnI enzyme, and the DpnI-treated PCR products were transformed into DMT chemically competent cells (Transgen Biotech). All mutants were verified by DNA sequencing. Positive colonies for the desired substitutions were grown in LB broth and the plasmids were isolated using an EndoFree mini plasmid DNA purification kit (Tiangen Biotech).