Gene Cloning and Mutagenesis
The gene fragment encoding mature SeMac was cloned from SEM1 with primers 5’-ACCATGGACGA TTACCAAAGGAATGCTAC-3’ and 5’-CGAATTCT TAGCTCAGTTTCTGCCATATG-3’. The protein made from this cloned fragment lacks the presumed secretion signal sequence (amino acids 1-34). The PCR product was digested with NcoI and EcoRI and ligated into pET-21d (Novagen) to yield recombinant plasmid pSEMAC. The cloned gene was sequenced and had identical DNA sequence with the corresponding open reading frame of S. equi genome database [12]. Amino acid replacement of Cys102 and His272 or Asp294 of SeMac with serine and alanine, respectively, was achieved by site-directed mutagenesis using the Quick-Change Mutagenesis kit (Stratagene, La Jolla, Calif.). The entire mutated gene was sequenced to confirm the mutations and rule out spurious mutations.