Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris.
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 P. 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 to ∼8mg/L. Biotinylated glycosylated and non-N-glycosylated soluble porcine CTLA-4 both bind to a porcine CD80-expressing B-cell lymphoma cell line (K(D)=13nM) 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.
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
Jaclyn
J
Peraino
Huiping
H
Zhang
Christina E
CE
Hermanrud
Guoying
G
Li
David H
DH
Sachs
Christene A
CA
Huang
Zhirui
Z
Wang
D000818:Animals; D018122:B7-1 Antigen / Q000378:metabolism; D060908:CTLA-4 Antigen / Q000096:biosynthesis* / Q000302:isolation & purification; D045744:Cell Line, Tumor; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D010843:Pichia; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012995:Solubility; D034421:Sus scrofa
D018122:B7-1 Antigen; D060908:CTLA-4 Antigen; D006023:Glycoproteins; D011994:Recombinant Proteins