A transient tobacco expression system coupled to MALDI‐TOF‐MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants

Abstract

Tobacco-based transient expression was employed to elucidate the impact of di¡erential targeting to subcellular compartments on activity and quality of gastric lipase as a model for the production of recombinant glycoproteins in plants. Overall N-linked glycan structures of recombinant lipase were analyzed and for the ¢rst time sugar structures of its four individual N-glycosylation sites were determined in situ by matrix-assisted laser desorption/ionization time-of-£ight mass spectrometry (MALDI-TOF-MS) on a trypsin digest without isolation or deglycosylation of the peptides. Three glycosylation sites contain both complex-type N-glycans and high-mannose-type structures, the fourth is exclusively linked to high-mannose glycans. Although the overall pattern of glycan structures is in£uenced by the targeting, our results show that the type of glycans found linked to a given Asn residue is largely in£uenced by the physico-chemical environment of the site. The transient tobacco system combined with MALDI-TOF-MS appears to be a useful tool for the evaluation of glycoprotein production in plants. ß

Characterization of the Novel CMT Enzyme TEM-154

“…TEM-154 was the 10th member of the complex mutant sub- (13,(15)(16)(17)(18)(19)(20). Such efficiency was similar to those of ESBL ceftazidimases such as TEM-6 and TEM-28 (k cat /K m , 0.4 and 0.27 M Ϫ1 ⅐ s Ϫ1 ) (18,19).…”

“…The purified proteins were resuspended in 5 mM ammonium hydrogen carbonate. Sinapinic acid was used as a matrix, and mass was measured using a peptide mixture (trypsinogen, enolase, bovine serum albumin; LaserBio Labs, Sophia-Antipolis, France) as an external standard as previously described (13). MALDI-TOF MS values (28,882.0 Da, 28,830.6 Da, and 28,815.6 Da) were in excellent agreement with the calculated molecular masses of the mature proteins (28,888.9 Da, 28,837.9 Da, and 28,819.9 Da, respectively).…”

Production and characterization of in planta transiently produced polygalacturanase from Aspergillus nigerand its fusions with hydrophobin or ELP tags

“…This result is consistent with at least two other CWDEs produced in sugar cane, where activities of cellobiohydrolase I (CBH I) and CBH II were, respectively, five and two times more active when targeted to the vacuole [34] when compared with ER-targeted proteins. As well, the effect of subcellular targeting on protein activity has been observed with human gastric lipase produced in tobacco; however, in this case, the vacuolar-targeted enzyme exhibited two-fold lower specific activity compared with the same protein targeted to the apoplast or ER, probably due to proteolytic-induced instability of this specific protein when targeted to the vacuole [35]. In the case of glycosyl hydrolases, higher specific activity was expected when targeted to the vacuole, as proteomic analysis of the vegetative vacuole of Arabidopsis thaliana uncovered several glycosyl hydrolases, including a polygalacturonase [36], which indicates that the vacuole is a natural compartment for these enzymes and may provide a chemical environment that favors appropriate protein folding and activity.…”

An efficient system to detect protein ubiquitination by agroinfiltration inNicotiana benthamiana

“…Transient expression has been described in several different species of plants. Among them, Nicotiana benthamiana is commonly utilized (Mokrzycki‐Issartel et al. , 2003; Wroblewski et al.…”

“…Transient expression has been described in several different species of plants. Among them, Nicotiana benthamiana is commonly utilized (Mokrzycki-Issartel et al, 2003;Wroblewski et al, 2005;Chakrabarty et al, 2007). There are several advantages to using N. benthamiana for transient protein expression.…”