Abstract
In part I, high efficiency transformation by electroporation is applied to yeast twon- hybrid screening. JHD1/YER051W is a demethylase gene from Saccharomyces cerevisiae BY4742. The JHD1/YER051W ORF is ligated to the BD vector as the “bait”. Appropriate DNA library constructed on the AD vector was screened for “fish” when the library was transformed into yeast PJ69-4A. The interacting proteins expressed from the AD vector would be selected in the specific medium. Afterwards, more functional analysis will be done to further understand the functional role of the interacting proteins. In this study, JHD1 was found to interact with CIN5/YOR028C gene product using the yeast two-hybrid screening. In part II, a nattokinase from Bacillus subtilis TKU007 was investigated. Propeptide, which is 77 amino acids fragment at the N terminus of the nattokinase, is required to convert pronattokinase to the mature active nattokinase. The propeptide is then cleaved off by the mature nattokinase. In this research, we used PCR (polymerase chain reaction) to clone pronattokinase and nattokinase sequence, respectively. Both of them are linked to His•Tag coding sequence at their C termini, and ligated to the expression vector pYES2. Heterologous expression in yeast was induced by galactose, and the over-expressed protein was purified .
Published Version
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