Expression of recombinant human IL-4 in Pichia pastoris and relationship between its glycosylation and biological activity
Introduction
Glycosylation is ubiquitous and essential protein post/co-translational modifications in eukaryotic cells [1]. Glycan structures play biological roles in protein folding, protein stability, enzymatic function, inflammation, cancer, and immunity [2]. Glycosylation increases the heterogeneity and functional diversity of proteins [3].
There are three major types of glycosylation: (1) the carbohydrate is added to a carbon on a tryptophan side chain; (2) the formation of a glycosylphosphatidylinositol (GPI)1 anchors (glypiation); and (3) phospho-linked, where the sugar is attached via the phosphate of a phospho-serine. Another classification system for protein-bound glycans is to describe them as N- or O-linked depending on the specific site of attachment. Great interest has been directed to N-glycosylation due to its essential role in proper protein folding. N-linked glycosylation, in eukaryotes, is catalyzed by a membrane associated enzyme oligosaccharyl transferase (OT) [4], a protein complex localized in the lumen of the endoplasmic reticulum (ER). N-linked glycosylation occurs at the Asn-Xaa-Ser/Thr sequon and is a co-translational process [5]. After OT-catalyzed glycosylation, Glc3MAN9GlcNAc2 is transferred to the polypeptide chain, glycosidase I and II trim the two terminal glucose residues, and the protein folding cycle begins. This pathway in eukaryotes has been described in detail [6], [7], [8]. For many higher eukaryotes, such as in mammalian systems, protein N-glycosylation is a highly coordinated and complex process. A plethora of diverse monosaccharide units are added to glycoproteins, such as GalNAc, GlcNAc, galactose, fructose, and N-acetylneuraminic acid [6], [9].
It has been relatively difficult to study glycoproteins because the glycan moieties usually have a heterogeneous chemical structure and conformation. Pichia pastoris, as a cellular host for the expression of recombinant protein, has become increasingly popular to use in recent times. Subsequent processing of newly glycosylated proteins occurs differently in P. pastoris compared with higher eukaryotic cells [10], however, P. pastoris is also a eukaryote, and thereby, provides the potential for producing soluble, correctly folded recombinant proteins [11] that have undergone the post-translational modifications required for functionality [12]. Additionally, P. pastoris is easier to genetically manipulate and culture than mammalian cells and this system is more efficient for production of recombinant proteins [13].
Human IL-4 (hIL4) consists of 153 amino acids that is synthesized as a precursor containing a hydrophobic secretory signal sequence of 24 amino acids and contains six cysteine residues, with the potential to form three disulfide bonds [14], [15]. The mature hIL4 contains 129 amino acids, including two potential N-glycosylation sites at Asn38 and Asn105 [16]. hIL4 is a 17 kDa monomeric glycoprotein secreted by T helper 2 (Th2) cells, NK T cells, mast cells, and basophils [17], [18], [19], [20] as an immunoregulatory cytokine [21]. hIL4 is involved in various physiological and pathological conditions, especially atopic diseases such as allergic hypersensitivity and asthma [22], [23], [24], bone loss [25], [26], and tumor immunology [27]. Blocking the activity of hIL4 might provide new therapeutic approaches to control allergies and inflammation [20].
Section snippets
Construction of an expression plasmid
The human IL4 cDNA (129 amino acids) was amplified by PCR on a plasmid encoding full-length human IL4 cDNA using the primers 5′TCTCTCGAGAAAAGACACAAGTGCGATATCAC3′ and 5′GATTCTAGATCAGCTCGAACACTTTGAA3′. PCR products were purified using a PCR purification kit (Invitrogen USA). The amplified PCR DNA was analyzed and confirmed by DNA sequencing. After cloning the PCR DNA into Xho1/Xba1 site of pPICZαA (Invitrogen,USA), the resulting plasmid pPICZα-IL4 was transformed into Escherichia coli (TOP10F)
Expression of recombinant hIL4 from P. pastoris
The time-course expression of hIL4 in yeast is shown in Fig. 1. After induction with 0.5% methanol, supernatants were collected at indicated time points by centrifugation. The harvested supernatants were subjected to SDS–PAGE and Western blotting assays (Fig. 1A, B). Recombinant hIL4 (rhIL4) was successfully expressed in our optimized culture conditions in a time-dependent manner. The rhIL4 produced in P. pastoris appeared as a broad smear band following SDS–PAGE and Western blot analysis (Fig.
Discussion
Biologically active rhIL4 protein was successfully expressed in P. pastoris yeast with a yield of 15.0 mg/L. The P. pastoris yeast expression system is a useful experimental tool for heterogeneous protein production. In previous studies, soluble hIL4 was produced constitutively in E. coli [28], [29]. However, human IL4 is a glycoprotein that participates in physiological and pathological processes in the body, and to our knowledge, the fully glycosylated hIL4 had not been produced so far. It is
Funding
This study was supported by Shandong University, PR China.
Acknowledgments
We would like to thank Dr. Jinghua Yang for LC–MS/MS analysis. We also are indebted to Dr. Xiangdong Wang for much helpful discussion and information regarding the Pichia pastoris protein expression system.
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