Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris. Proteins. Genes & Expression. Gene.

The publisher's final edited version of this article is available at Glycoconj J

Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance.

We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections.

Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF.

Compared to the mammalian cell culture technology currently in use for the production of most therapeutic glycoproteins, the methylotrophic yeast Pichia pastoris has the capability of yielding up to 20 times more product.

The complex metabolic engineering endeavor of replicating the mammalian glycosylation machinery in yeast requires the cloning and functional expression of a large number of foreign glycosylation pathway enzymes in the host strains. Each enzyme catalyzes a reaction yielding the substrate for the subsequent enzyme. Thus, each enzyme must be properly targeted and must function at high efficiency in its respective location in the secretory pathway.

It is considered a first line defense protein involved in protection against microbial infections [ 26 , 31 ] and prevention of systemic inflammation [ 2 , 3 , 21 ]. More recently, lactoferrin has been implicated in immunoregulatory functions [ 19 , 42 , 44 , 46 ], as a modulator of vaccine function [ 18 ], and containing chemoprotective activity [ 1 ]. The primary structure of human LF is characterized by a single polypeptide chain containing 692 amino acids organized in two highly homologous lobes, designated the N- and C-lobe, each capable of binding one ferric ion (Fe+++).

The low-density lipoprotein receptor-related protein-1 and -2 (LRP1 and LRP2) are considered primary LF receptors. Although members of the LRP family are generally considered as endocytic receptors, LRP1 can also function as a signaling receptor [ 29 ]. The key to understanding the molecular basis of LF various activities is thought to reside in part according to patterns of glycosylation [ 37 ].

There are three possible N-linked glycosylation sites in hLF, one at Asn138, a second site at Asn479, and a third site at Asn624; differential utilization of these sites results in distinct glycosylation variants [ 30 , 35 ]. Human LF glycans are the N-acetyllactosaminic type, &x003b1;1-3-fucosylated on the N-acetylglucosamine residue linked to the peptide chain.

Strains, culture conditions and reagents Escherichia coli strain TOP10 was used for recombinant DNA work. P. pastoris yAS309 [ 24 ] was used for generation of rhLF producing strains. Protein expression studies were done at room temperature in a 96-well plate format with buffered glycerol-complex medium (BMGY) consisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base, 4&x000d7;10&x02212;5% biotin, and 1% glycerol as a growth medium.

The induction media were buffered methanol-complex medium (BMMY) consisting of 0.5% methanol and buffered dextrose-complex medium (BMDY) consisting of 2% dextrose, respectively. hLF standard purified from human milk was purchased from Sigma (St. Louis, MO).

For the signal sequence study, pPICZA (Invitrogen, Carlsbad, CA) was digested with EcoRI and KpnI, and the resulting pPICZA was ligated with 11 different signal sequences (EcoRI and blunt ended) and the codon-optimized hLF cDNA (blunt and KpnI ended) (provided by PharmaReview Corporation, Houston, TX). For the promoter study, PpAOX1 promoter was replaced with the PpGAPDH promoter in pBK422 at BglII and EcoRI sites.

All expression constructs were sequence verified.

A seed culture was prepared by adding 1 ml of thawed cells to a 2 L baffled flask containing 400 ml of 4% BMGY medium.

Primary clarification was performed at 4&x000b0;C for 15 min at 13,000&x000d7;g by centrifugation in a Sorvall Evolution RC (Kendo, Asheville, NC) followed by the microfiltration and diafiltration steps using a 0.1 &x003bc;m cut-off 3600 cm2 PES hollow fibre cartridge (CFP-1-E-8A) (GE Amersham, Pittsburgh, PA) and 5&x000d7;0.1 m2 Pellicon 2 Mini 10 kDa NMWCO regenerated cellulose ultrafiltration cassettes (A screen) (Millipore, Billerica, MA), respectively.

Protease inhibitors pepstatin A and chymostatin (Sigma) were added to the supernatant after filtration steps in a concentration of 5 and 3 &x003bc;g/ml respectively. After the ultrafiltration/diafiltration/filtration steps, P. pastoris-derived rhLF was purified by two chromatographic steps; cation exchange chromatography using SP Sepharose Fast Flow followed by Heparin Sepharose 6 Fast Flow chromatography (GE Healthcare, Piscataway, NJ).

Briefly, SP Sepharose resin was equilibrated with 50 mM Tris&x02013;HCl, pH 8.0 while the supernatant media was adjusted at the same pH and conductivity around 5 mS/cm by the diafiltration procedure. The elution was done with 10 column volume (CV) of a gradient of 0&x02013;1 M NaCl in the same buffer. rhLF containing fractions were pooled and dialyzed against 50 mM Tris&x02013;HCl, pH 7.5 overnight.

As the final purification step the affinity resin, Heparin Sepharose 6 Fast Flow, was used and the column was equilibrated with 50 mM Tris&x02013;HCl (pH 7.5). The pooled and dialyzed protein from SP Sepharose was loaded on Heparin Sepharose and washed in three steps. The first wash was done with 2 CV of the same buffer, followed by the second wash with 10 CV of a detergent buffer (10 mM CHAPS, 10 mM EDTA in 50 mM Tris&x02013;HCl, pH 7.5) to decrease endotoxin levels.

The last wash was carried out with a 10 CV of 50 mM Tris&x02013;HCl (pH 7.5). The protein was eluted with a 10 CV of a gradient of 0&x02013;1 M NaCl.

Mice were primed with intraperitoneal administration of 0.2 ml 1% sheep red blood cells (SRBC) suspension. Splenocytes were isolated after 4 days and single cell suspensions were prepared in culture medium consisting of RPMI 1640, supplemented with 10% fetal calf serum, glutamine, sodium pyruvate, 2-mercaptoethanol and antibiotics. The cells were incubated in 24-well culture plates (5&x000d7;106/ml/well) with 50 &x003bc;l 0.005% SRBC.

LF could prevent MTX-induced suppression in several ways, including the induction of specific cytokines in sialoadhesin-positive splenocytes. Mediators such as IL-6 [ 33 ], IL-12 [ 39 ], and TGF-beta [ 7 ] induce anti-apoptotic responses and LF indeed increases the production of such mediators [ 11 , 19 , 41 ].

28. Nakamura K, Yamaji T, Crocker PR, Suzuki A, Hashimoto Y. Lymph node macrophages, but not spleen macrophages, express high levels of unmasked sialoadhesin: implication for the adhesive properties of macrophages in vivo.

30. Samyn-Petit B, Wajda Dubos JP, Chirat F, Coddeville B, Demaizieres G, Farrer S, Slomianny MC, Theisen M, Delannoy P. Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants. Eur. J. Biochem.

[ PMC free article ] [ PubMed ] .. . 32. Spik G, Coddeville B, Montreuil J. Comparative study of the primary structures of sero-, lacto- and ovotransferrin glycans from different species. Biochimie. 1988;70 :1459?1469. [ PubMed ] 33. Takeda K, Kaisho T, Yoshida N, Takeda J, Kishimoto T, Akira S. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice.

J. Immunol.

J.

The C-type lectin superfamily in the immune system.

Effects of lactoferrin on the immune response modified by the immobilization stress.

Lactoferrin (LF) lowers IgM and interleukin 2 receptor expression on WEHI 231 cells and decreases anti-IgM antibody-induced cell death. Pol. J. 1995. p. 324.

[Nat Biotechnol. 2004]. See more articles cited in this paragraph .. Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol. 2006 Feb; 24(2):210-5 [Nat Biotechnol. 2006] Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad Sci U S A. [Proc Natl Acad Sci U S A.

Review Lactoferrin and the inflammatory response.

Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice.

Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris.

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.

Immunization of dissociated spleen cell cultures from normal mice.

Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris. Protein Expr Purif. [Protein Expr Purif. Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin: a study with glycosylation-site mutants. Biochem J.

Production of complex human glycoproteins in yeast.

2003] Humanization of yeast to produce complex terminally sialylated glycoproteins.

Biochem J.

Comparative study of the primary structures of sero-, lacto- and ovotransferrin glycans from different species.

Review The C-type lectin superfamily in the immune system.

Purification and properties of sialoadhesin, a sialic acid-binding receptor of murine tissue macrophages.

A member of the dendritic cell family that enters B cell follicles and stimulates primary antibody responses identified by a mannose receptor fusion protein.

Proteins. Sequence Analysis. Genes & Expression. Domains & Structures. Data & Software. Small Molecules.

Gene.

Structure.

Human Genome.

Sequence Read Archive.