My name is Tita Foophow and I am currently a first year Ph.D. student at Osaka University in the Department of Material and Life Science under the Laboratory of Molecular Biotechnology of Prof. Shigenori Kanaya. I got the good opportunity to go to Eotvos Lorand University (ELTE), Hungary in the Department of Biochemistry under the Laboratory of Assoc. Prof. Istvan Venekei as a student internship during February 3rd to March 14th 2009. I did experiment on the construction and purification of SPH-3, serine protease homologue 3, in yeast cells. SPH-3 is one of the six identified substrate protein of PrtA and the function of SPH-3 is unknown. Therefore, they would like to construct SPH-3 in order to know more about its function. Unfortunately, SPH-3 failed to express in bacteria. Thus, it was constructed in a yeast expression system.
My experiment was preparing the plasmid DNA, transformation yeast cells, testing the expression with two different types of western blot, and purify the protein with affinity chromatography on Ni-agarose. My experiment found that SPH-3 was successfully expressed in the culture supernatant, which it was checked the expression using western blot (immunoblot). It indicated that SPH-3 was overproduced and secreted as an extracellular protein from the yeast cells. For purification of SPH-3, the culture supernatant after centrifugation was precipitated by adding 50% ammonium sulfate and applied to affinity chromatography on Ni-agarose. The purified protein will be used as a bait to fish for interacting protein to SPH-3 in Manduca hemolymph for study the function of its.
I gain the grate experience and learn laboratory techniques about the yeast expression system in part of construction, transformation, expression, and immunoblot for expression check, which I never learn before from our lab. These experiences will help me to solve the production level of Pro-Tk-SP, a subtilisin like-serine protease from Hyperthermophilic Archaeon, Thermococcus kodakaraensis (my project) because the production level of Pro-Tk-SP, which was over-expressed in bacteria, is too low. If we can increase the production yield of protein, it will useful for the production in industrial field by decrease the time and cost of production process. Normally, subtilisin has been widely used for industrial purposes mainly as additive of cleaning agents which, the application of Pro-Tk-SP in industrial fields is one of the objectives of my project. Therefore, the yeast expression system is a probably alternative way to increase the production level of protein.
With the grate opportunity, I am not only gain the good experience for laboratory techniques but also have a good chance to know and discuss with other people, who are studying and interested in the related field with mine and spend the time to learn the new culture and life-style such as characteristic, sociality, religion, food, and travel in Europe country, which is quite different from Asian country.