T. brucei GII and mutanase tested as engineering approach.
(A) The dual N-glycosylation system in T. brucei. Both Man9GlcNAc2 and Man5GlcNAc2 can be transferred to proteins. Next, these proteins are reglucosylated and deglucosylated in the folding cycle by glucosyltransferase and GII, respectively. (B) DSA-FACE analysis of reference N-glycans and N-glycans derived from strains engineered with T. brucei GII or treated with mutanase. A, Oligomaltose reference. B, N-glycans from RNaseB reference. C, N-glycans from the alg3 mutant strain overexpressing Alg6p. D-F, N-glycan from the alg3 mutant strain overexpressing Alg6p and engineered in different ways: D, engineered with T. brucei GII; E, engineered with T. brucei GII with HDEL tag; F, engineered with T. brucei GII with HDEL tag and pre-lip2 signal. G, N-glycans derived from the alg3 mutant strain overexpressing Alg6p treated with mutanase.