Investigation of an Elatin-like Polypeptide Tag Length
MetadataShow full item record
Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Chemical and Biomolecular Engineering Honors Theses; 2011
Recombinant protein technology can be used to express a chimeric protein fusion in prokaryotic cells to yield a purified single protein product. By using the elastin-like polypeptide (ELP) affinity tag technology in conjunction with a self-cleaving protein domain from a bacterial intein element, the target protein can be purified using only a change in salt concentration and pH. Shortening the affinity-purification peptide tag has the potential to increase expression by freeing more of the cells energy to produce the valuable protein product. The reduction in tag length has the potential of making this novel purification system more industrially applicable by increasing production of the desired product and eliminating the need for costly chromatography columns. The affinity tag length needed may be affected by the size and solubility of the protein product fused with the tag. For this study, different tag sizes from 250 up to the original 550 amino acid length were cloned into pET vectors with two separate target proteins of 255 and 1,024 amino acids long. The pET vectors were expressed in BLR E. coli cells and purified using 0.4 M ammonium sulfate. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to qualitatively determine the success of the purification. Activity and Bradford assays were then used to follow quantitatively the success of the purification. A minimum of a 27 % reduction in tag length was shown to uphold successful purification, and was dependent on the fused protein. Target protein size however was shown not to be the dominating factor in precipitation and success of the purification. More proteins should be looked at to develop a correlation for tag length based on other factors such as hydrophobicity.
1st Place in Engineering at 2011 Denman Undergraduate Research Forum
Items in Knowledge Bank are protected by copyright, with all rights reserved, unless otherwise indicated.