Characterization of recombinant human lactoferrin expressed in Komagataella phaffii

Abstract

This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

Fig. 1.

The three types of N-glycans that are present in the glycoproteins of both mammals and yeast, namely complex, hybrid, and oligomannose structures (A). The N-glycans share a common core Man3GlcNAc2 which is linked to the Asparagine (Asn) residue of the glycoproteins having Asparagine (Asn)-X-Serine (Ser)/Threonine (Thr) sequons. Unlike mammals, the yeast tends to synthesize hypermannose N-Glycans (B). The colored shapes represent monosaccharide residues: ■ N-acetylglucosamine (GlcNAc); ● mannose (Man); ● galactose (Gal), ▲ fucose (Fuc); and ◆ N-acetylneuraminic acid (NeuAc).

Fig. 2(A).

Protein sequence of native hmLF (Uniprot P02788) without the N-terminal signal peptide (amino acid sequence 1–19). The highlighted N indicates the three known N-glycosylation sites, namely, Asn-156, −497 and −642; (B). Detected sequence of native hmLF from Helaina rhLF as highlighted by the yellow and green color. The green color indicates posttranslational modifications.

Fig. 3.

Representative images of total protein assay and Simple Western^™ of native hmLF (Lane-2) and Helaina rhLF (Lane-3) at the concentration of 100 μg/mL. Other conditions are described in Materials and Methods.

Fig. 4.

Representative chromatograms of RP-HPLC analysis of Helaina rhLF with UV detection at 280 nm (A) and 214 nm (B). * Indicates impurity peaks observed from the rhLF samples. The chromatograms were generated from 1.0 μL injection of 1.0 mg/mL Helaina rhLF. Other conditions are described in Materials and Methods.

Fig. 5.

Comparison of the released N-glycan profiles of Helaina rhLF (A) and bLF (B). Conditions for data acquisition and processing are detailed in the Material and Methods. Fig. 5. The N-glycan profiles of Helaina rhLF (A) and bovine LF (B)

Fig. 6.

Representative LC-fluorescence chromatogram of the released N-glycan profile of native hmLF. The peak assignments were supported by a LC-HRIM-MS trace with good alignment between the peaks. Each of the peaks is labeled with the m/z value(s) of the most abundant species observed and the best possibly matched N-glycan(s). The two red circles indicate the detection of oligomannoses M5 and M6, which are also present in Helaina rhLF. Other conditions are described in Materials and Methods.

Fig. 7.

Representative second derivative MMS spectra of Helaina rhLF, native hmLF and native bLF (A); and comparison of the compositions of the secondary structures of Helaina rhLF with native hmLF (B). The composition of the secondary structures of Helaina rhLF was the average from three production batches, while that of native hmLF was the average from three different sources of hmLF as described in the Material section.

Fig. 8.

Representative solvent envelopes of Helaina rhLF and native hmLF derived from the SAXS data and their fits with the Fe⁺³ bound human lactoferrin monomer (2bjj) model. Conditions for data acquisition and processing are detailed in the Material and Methods.

Fig. 9.

Comparison of the radii of gyration (R_g) of the Helaina rhLF proteins with native hmLF proteins. Conditions for data acquisition and processing are detailed in the Material and Methods.

Fig. 10.

AUC measurements of native hmLF (A) and Helain rhLF (B). The data presented a pseudo-three-dimensional distribution of the observed protein species, with the calculated molecular weight on the x-axis, the calculated frictional ratio on the y-axis, and the % concentration in the Z-plane, with the heat map on the right axis. Conditions for data acquisition and processing are detailed in the Material and Methods.

Fig. 11.

Comparison of differential scanning calorimetry of native hmLF (apo- and holo-form) (A) and Helaina rhLF (B). Conditions for data acquisition and processing are detailed in the Material and Methods.