Construction of the OPHC2
KDS-4103 vector. The OPHC2 expression vector was constructed according to standard protocols [25]. The gene encoding OPHC2, ophc2, was amplified by PCR from the genomic DNA of P. pseudoalcaligenes C2-1 using primer_F (TACGTA ACC ATG GCC GCA CCG GCA CAA CAG AAG) and primer_R (GAGCTC TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT ATC AGC GGT CGC TAC GGA TCG G). The underlined sequences correspond to SnaBI and SacI sites, respectively. The PCR product was purified using the MinElute? PCR purification kit (QIAGEN, USA), digested with SnaBI and SacI, and directionally cloned into the SnaBI and SacI restriction sites of the vector Teasy-ex, which contained a signal peptide sequence from the carrot extensin gene [26] and [27]. The recombinant vector was designated pTeasy-ophc2. The extensin signal sequence facilitated the extracellular
expression of OPHC2. The ophc2 with the extensin sequence was excised using SacI and Xbal from pTeasy-ophc2 and ligated into pBI121. The final expression cassette, which contained the cauliflower mosaic virus (CaMV) 35S promoter, the extensin sequence, ophc2, and the nos termination sequence, was designated pBI-E-ophc2.
