Screening of Streptavidin Affinity Peptides

• Main Authors: Huang Tsun-Han, 黃遵韓
• Other Authors: Chen Hsiu-mei
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/37006716085893296762

碩士 === 國立臺灣科技大學 === 化學工程系 === 90 === This thesis reports the combination use of a novel screening strategy with new screening tools to find peptides with high affinity to streptavidin (SAv). First, a random deca-peptide library displayed on the flagellum of E.coli was constructed. Second, the library was pre-screened for the colonies with SAv binding affinity by using avidin-coated glass slides, which were prepared by hydrogel coating. The prescreened colonies were then re-screened with fluorescent-labeled SAv. Finally, an SAv pizeoelectric (PZ) biosensor was used to analyze the binding kinetics of SAv with previously screened colonies and consequently determined the colonies displaying the peptides of highest SAv affinity based on their kinetic parameters. The novel strategy used in the screening process was to avoid over-amplification of E. coli cells before each screening step by analyzing the growth curve. The results showed that the random peptide library was successfully constructed with the diversity near 100% which was determined by DNA sequencing and that 2880 clones out of 1011 library were screened out with avidin —coated slides. The microarray experiment further reduced to the screened library size down to 53 clones that showed twice higher fluorescence intensities compared to that of the negative control one, a clone carrying the nascent plasmid without the insertion of any random peptide gene. Two clones out of 53 were assayed with a SAv-PZ biosensor and the BNo. 23 one showed a higher SAv affinity than the other one. In addition, the affinity value of the BNo. 23 clone was close to that of the clone carrying strep II peptide that was already known to have SAv affinity. This study showed the feasibility of the combination of the novel screening strategy with biopanning, microarray and biosensor tools to screen for an SAv affinity peptide from a flagellum displayed library.