Expression and isolation of the membrane proteoglycan syndecan-1 from E. coli

Abstract

Syndecan-1 is a membrane proteoglycan, which is a protein with glycosaminoglycans, or long carbohydrate chains, attached to its extracellular domain. The syndecan family is composed of various proteoglycans that each aid in cell-to-cell communication, and cell signaling pathways. Syndecan-1 plays a role in the initiation and progression of many different types of cancer. The overall goal of our research is to express and isolate the human syndecan-1 protein in bacterial cells, and then proceed with glycosylation of the protein. In order to have effective expression and cleavage of syndecan-1, a His6-TrpΔLE fusion partner was attached to the syndecan-1 protein, and the sequence of syndecan-1 was altered to replace any cysteine and methionine residues with serine residues. The path to achieving this involves a growth of cells with emphasis on the expression of syndecan-1, a chemical cleavage of the TrpΔLE fusion partner from the syndecan-1 protein, purification by size-exclusion chromatography, and each step monitored by SDS-PAGE to show expression and purity. The syndecan-1 protein in its altered form weighs 31.903kDa, and the TrpΔLE weighs approximately 14kDa. We found that we were successful in cleavage and expression via SDS-PAGE, and got separation of protein through size-exclusion chromatography, but have not expressed a large amount of protein from growths, nor have we gotten proof of our protein expression by mass spectrometry. In the future, we plan on making slight adjustments in protocol and doing more growths to create more protein to work with, and find a reason that we are unable to identify the protein by mass spectrometry.