Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris

Abstract

Co-stimulation blockade can be used to modulate the immune response for induction of organ transplantation tolerance, treatment of autoimmune disease as well as cancer treatment. Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), also known as CD152, is an important co-stimulatory molecule which serves as a negative regulator for T cell proliferation and differentiation. CTLA-4/CD28-CD80/CD86 pathway is a critical co-stimulatory pathway for adaptive immune response. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for CD80 and CD86. MGH MHC-defined miniature swine provide a unique large animal model useful for preclinical studies of transplantation tolerance and immune regulation. In this study, we have expressed the codon-optimized soluble porcine CTLA-4 in the yeast Pichia pastoris system. The secreted porcine CTLA-4 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50HQ. Glycosylation analysis using PNGase F demonstrated the N-linked glycosylation on Pichia pastoris expressed soluble porcine CTLA-4. To improve the expression level and facilitate the downstream purification we mutated the two potential N-linked glycosylation sites with non-polarized alanines by site-directed mutagenesis. Removal of the two N-glycosylation sites significantly improved the production level from ~2 mg/L to ~8 mg/L. Biotinylated glycosylated and non-N-glycosylated soluble porcine CTLA-4 both bind to a porcine CD80-expressing B-cell lymphoma cell line (KD = 13 nM) and competitively inhibit the binding of an anti-CD80 monoclonal antibody. The availability of soluble porcine CTLA-4, especially the non-N-glycosylated CTLA-4, will provide a very valuable tool for assessing co-stimulatory blockade treatment for translational studies in the clinically relevant porcine model.

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

“…As shown here, this is indeed the case for the antibacterial enzyme LST. In most instances, destruction of the glycosylation sequon is accomplished by replacing the N-linked asparagine residue with glutamine, aspartic acid, serine, or alanine (40)(41)(42)(43). In the current study of LST glycosylation, all mutations at the Nlinked N125 residue led to significantly decreased enzyme activity, whereas mutations at two adjacent residues (S126 and T127) yielded fully functional aglycosylated variants.…”

Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25+ cells

“…2) was synthesized by PCR amplification as described previously (Hermanrud et al, 2011). To construct DT390-hIL-2, the codon-optimized human IL-2 DNA was amplified using PCR primers hIL2-X1 carrying Xho I and Nco I site + hIL2 Rev carrying an Eco RI site then cloned into pwPICZalpha (Peraino et al, 2012a) between Xho I and Eco RI sites for sequencing confirmation. The insert was then cut out with Nco I + Eco RI and cloned into pwPICZalpha-DT390 (Wang et al, 2011) between Nco I and Eco RI sites yielding the final construct DT390-hIL-2 in pwPICZalpha.…”

Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells

“…The codon-optimized glycosylated porcine IL-2 DNA was synthesized by GenScript (Piscataway, NJ) and the codon-optimized non- N -Glycosylated porcine IL-2 DNA was generated by site-directed mutagenesis with sense PCR primer pIL2-N91A For and anti-sense PCR primer pIL2-N91A Rev (Agilent technologies). To construct DT390-pIL-2-Gly or DT390-pIL-2-Non- N -Gly, the codon-optimized glycosylated or non- N -glycosylated porcine IL-2 DNA (Figure 2) was amplified using PCR primers pIL2-X1 carrying Xho I and Nco I site + pIL2-E1 carrying an Eco RI site then cloned into pwPICZalpha (Peraino et al, 2012) between Xho I and Eco RI sites for sequencing confirmation. The insert was then cut out with Nco I + Eco RI and cloned into pwPICZalpha-DT390 (Wang et al, 2011) between Nco I and Eco RI sites yielding the final construct DT390-pIL-2-Gly or DT390-pIL-2-Non- N -Gly in pwPICZalpha.…”

“…Protein expression and purification in Pichia pastoris were performed as previously described with the following modifications (Wang et al, 2011; Peraino et al, 2012). A Ni-Sepharose fast flow resin (GE healthcare) was used for the purification.…”

“…Porcine IL-2 fusion toxins were eluted using 40 mM imidazole. Western blot analysis, FACS analysis, FACS competition/blocking analysis and K D determination were all performed as previously described (Peraino et al, 2012) using LCL13271 cells (Cho et al, 2007). The Ontak ® -like monovalent human IL-2 fusion toxin (DT390-hIL-2) used as a control for our in vitro assay was constructed, expressed and purified exactly same as the monovalent porcine IL-2 fusion toxin.…”