Fusion of a highly N-glycosylated polypeptide increases the expression of ER-localized proteins in plants

Abstract

Plants represent promising systems for producing various recombinant proteins. One key area of focus for improving this technology is developing methods for producing recombinant proteins at high levels. Many methods have been developed to increase the transcript levels of recombinant genes. However, methods for increasing protein production involving steps downstream of transcription, including translation, have not been fully explored. Here, we investigated the effects of N-glycosylation on protein production and provide evidence that N-glycosylation greatly increases the expression levels of ER-targeted recombinant proteins. Fusion of the extracellular domain (M domain) of protein tyrosine phosphatase receptor type C (CD45), which contains four putative N-glycosylation sites to a model protein, leptin at the C-terminus, increased recombinant protein levels by 6.1 fold. This increase was specific to ER-targeted proteins and was dependent on N-glycosylation. Moreover, expression levels of leptin, leukemia inhibitory factor and GFP were also greatly increased by fusion of M domain at either the N or C-terminus. Furthermore, the increase in protein levels resulted from enhanced translation, but not transcription. Based on these results, we propose that fusing a small domain containing N-glycosylation sites to target proteins is a powerful technique for increasing the expression levels of recombinant proteins in plants.

Figure 1

Amino acid sequence of the M domain. The amino acid sequence of human CD45 from Ala231 to Asp290 is shown. The putative N-glycosylation sites of the four Asn residues are indicated by N1 to N4.

Figure 2

Fusion of the M domain increases the expression levels of recombinant protein. (a) Schematic representation of the constructs. All target sequences were expressed under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and heat shock protein (HSP) terminator. ER-targeted proteins contain the leader sequence of BiP. Recombinant proteins targeted to the chloroplast or mitochondria contain the transit peptide of Cab or the presequence of the F1-ATPase gamma subunit, respectively. Enk, enterokinase. (b) Western blot analysis of various recombinant proteins. Protein extracts from protoplasts transformed with the indicated constructs were analyzed by western blotting using anti-HA antibody. Actin (detected using anti-actin antibody) was used as a loading control. EeLepf and EeLepfM are ER-targeted proteins, CeLepf and CeLepfM are chloroplast-targeted proteins, and FeLepf and FeLepfM are mitochondria-targeted proteins. (c) Quantification of protein levels. The intensity of protein bands from Fig. 2(b) was quantified using the software provided with the LAS4000 image analyzer; values relative to that of ER-targeted EeLepf are shown. Error bars, standard deviation (n = 3). Means with different letters indicate significant difference (Tukey’s test P < 0.05). (d) Schematic representation of unglycosylated mutant protein M1234. Four Asn residues were substituted with Gln residues as indicated. (e) Analysis of N-glycosylation. Protoplasts transformed with the indicated constructs were incubated for 24 h with or without tunicamycin (10 μg/mL). Protein extracts from transformed protoplasts were analyzed by western blotting using anti-HA antibody. (f) Quantification of protein levels. To quantify protein levels, the signal intensity of each band in Fig. 2(e) was measured using the software provided with the LAS4000 system; values relative to that of tunicamycin-untreated EeLepf (EeLepf -) are shown. Three independent experiments were performed. Error bar, standard deviation (n = 3). Means with different letters indicate significant difference (Tukey’s test P < 0.05).

Figure 3

M domain fusion generally increases protein expression levels regardless of the position. (a) Schematic representation of constructs with or without the M domain. (G₄S)₂ indicates a peptide linker containing two copies of a sequence comprising four Gly and one Ser residue. Xxx is model protein including as leptin (Lep), LIF (Lif), GFP (Gfp). (b–d) Expression levels of fusion proteins. Protein extracts from transformed protoplasts with the indicated constructs were analyzed by western blotting using anti-HA (b,c) or anti-GFP antibodies (d). Actin was used as a loading control.

Figure 4

N-glycosylation-mediated increase in fusion protein levels is dependent on individual N-glycosylation sites in the M domain and their combination. (a–c) Expression levels of various Asn-to-Gln substitution mutants. The indicated Asn-to-Gln mutants were expressed in protoplasts. Protein extracts from transformed protoplasts were analyzed by western blotting using anti-HA antibody. Western blot images are shown in Fig. S1. The signal intensity of protein bands shown in Fig. S1 was quantified using software provided with the LAS4000 image analyzer; relative values to that of the ER-targeted wild-type protein, EeLepfM, are shown. (a) Relative levels of single Asn-to-Gln mutants; (b) double Asn-to-Gln mutants; (c) triple Asn-to-Gln mutants. Error bars, SD (n = 4). Numbers in these mutant constructs indicate Asn residues at the first to fourth position substituted with Gln. Means with different letters indicate significant difference (Tukey’s test P < 0.05).

Figure 5

Low expression levels of unglycosylated proteins are not caused by ER-associated degradation. (a) Scheme of experimental design. MG132 was added to the protoplast incubation medium at 18 or 21 h after transformation of the indicated constructs, and the protoplasts were further incubated for 6 or 3 h, respectively. HAT, h after transformation. (b) Western blot analysis of proteins. Protein extracts were prepared from protoplasts harvested at 24 h after transformation and analyzed by western blotting using anti-HA antibody. RbcS[T4,7 A]:GFP was analyzed using anti-GFP antibody as a positive control for 26S proteasome-mediated degradation and MG132 treatment. Actin (detected using anti-actin antibody) was used as a loading control.

Figure 6

The translation rate of the M domain-containing recombinant protein is higher than that of the mutant fusion protein containing an unglycosylated M domain. (a) Scheme of experimental design. Cycloheximide (CHX) or DMSO (as a control) was added to the protoplast incubation medium at 12 h after transformation of the indicated constructs, and protoplasts were harvested at the indicated time points. (b) Western blot analysis of protein levels. Protein extracts from protoplasts were analyzed by western blotting using anti-HA antibody. Actin (detected using anti-actin antibody) was used as a loading control. (c) Quantification of protein levels. The signal intensities of the protein bands in Fig. 6(b) were measured using software provided with the LAS4000 image analyzer and are expressed as relative values to that of the corresponding constructs in 12 HAT samples Error bars, SD (n = 3). Repeated-measures two-way analysis of variance (ANOVA) detected a significant expression level difference between EeLepfM and EeLepfM1234 in the absence (DMSO) or presence (CHX) of cycloheximide (*p < 0.05; ***p < 0.001).