The present invention provides methods of refolding mammalian glycosyltransferases that have been produced in bacterial cells, and methods to use such refolded glycosyltransferases, including glycosyltransferase mutants that have enhanced ability to be refolded. The invention also provides methods of refolding more than one glycosyltransferase in a single vessel, methods to use such refolded glycosyltransferases, and reaction mixtures comprising the refolded glycosyltransferases.