Purification of Sm1 from T. virens results in low protein yield limiting the application of this protein for crop disease protection to small-scale assays. To increase the yield see more of 51111, we cloned the sm1 gene in the pPIC9K vector for transformation into the AOX1 locus of Pichia pastoris strain GS115. Transformants

of P. pastoris were selected based on the presence of the vector insert as indicated by PCR analysis and the ability to secrete high levels of the rSm1 protein. The optimal incubation period and methanol concentrations for induction were determined for production of rSm1 in shake flasks. One Pichia transformant was estimated to express approximately 55 mg/l of rSm1 after 4 days culture in a 1% final concentration of methanol. The secreted rSm1 was purified by ammonium sulfate precipitation, ion exchange chromatography and gel column chromatography. SDS-PAGE and Western blot analysis revealed that the purified rSm1 expressed in Pichia was recognized by anti-Sm1 polyclonal antibody. The protein sequence was verified by

ESI/MS/MS analysis of a tryptic digest of the rSm1. Greater than 90% peptide coverage was obtained and determined to be identical to the predicted sequence. The MALDI/TOF/MS analysis revealed the molecular mass of rSm1 to be 13.1 kDa, which is higher than native Sm1 (12.6 kDa). Edman sequencing of the purified protein revealed an N-terminal extension of six amino acids (EAEAYV). The extension is the result of insufficient activity of BAY 63-2521 the Ste13 protease preventing efficient cleavage of the spacer (EAEA) downstream of the Kex2 cleavage site. Maize (cv. Silver Queen) treated with rSm1 or native Sm1 demonstrated the induction of two defense genes. Enhanced production of this elicitor has implications for the treatment of specialty crops to promote disease resistance. (C) 2010 Elsevier Inc. All rights

4��8C reserved.”
“Gastroesophageal reflux disease is the most common esophageal disorder encountered in the United States. Gastroesophageal reflux disease symptoms are associated with a negative quality of life and increased healthcare costs and therefore require an effective management strategy. Although proton pump inhibitors remain the primary treatment of gastroesophageal reflux disease, they do not cure the disorder and can leave patients with persistent symptoms despite treatment. Moreover, patients are still at risk of developing such complications as peptic strictures, Barrett’s metaplasia, and esophageal cancer. Although laparoscopic Nissen fundoplication has been the conventional alternative treatment for those patients who develop complications of gastroesophageal reflux disease, have intractable symptoms, or wish to discontinue taking proton pump inhibitors, investigators have persisted in developing a number of endoscopic approaches to the treatment of gastroesophageal reflux disease.