In Vitro Folding of β-1,4Galactosyltransferase and Polypeptide-α-N-Acetylgalactosaminyltransferase from the Inclusion Bodies

Abstract

The aim of this article is to present a unique in vitro folding technique for glycosyltransferases to generate active proteins that can be used for X-ray crystallographic and bioconjugation protocols. Although a number of in vitro refolding methods are available, β1,4galactosyltransferases in large quantities can be made using the current protocol only. This technique is not only limited to glycosyltransferases alone but has been successfully used to refold single-chain antibodies and other molecules. Although this in vitro folding method is quite similar to other methods, it differs from them by the use of S-sulfonation of the inclusion bodies before setting up the in vitro refolding of the protein.

Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation

“…The first step of the chemoenzymatic approach is the selective labeling of O-GlcNAcylated proteins with GalNAz. This is achieved using Y289L GalT, which is robustly expressed in E. coli , purified from inclusion bodies, and refolded to obtain active enzyme (Ramakrishnan & Qasba, 2013). The below procedure is a streamlined protocol for making large quantities of Y289L GalT.…”