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Protein Expr Purif. 2012 Mar;82(1):70-4. doi: 10.1016/j.pep.2011.11.011. Epub 2011 Dec 1.

Expression and purification of non-N-glycosylated porcine interleukin 3 in yeast Pichia pastoris.

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  • 1Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

Abstract

Yeast Pichia pastoris has been widely utilized to express heterologous recombinant proteins. P. pastoris expressed recombinant porcine interleukin 3 (IL3) has been used for porcine stem cell mobilization in allo-hematopoietic cell transplantation models and pig-to-primate xeno-hematopoietic cell transplantation models in our lab for many years. Since the yeast glycosylation mechanism is not exactly the same as those of other mammalian cells, P. pastoris expressed high-mannose glycoprotein porcine IL3 has been shown to result in a decreased serum half-life. Previously this was avoided by separation of the non-glycosylated porcine IL3 from the mixture of expressed glycosylated and non-glycosylated porcine IL3. However, this process was very inefficient and lead to a poor yield following purification. To overcome this problem, we engineered a non-N-glycosylated version of porcine IL3 by replacing the four potential N-glycosylation sites with four alanines. The codon-optimized non-N-glycosylated porcine IL3 gene was synthesized and expressed in P. pastoris. The expressed non-N-glycosylated porcine IL3 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50 HQ. In vivo mobilization studies performed in our research facility demonstrated that the non-N-glycosylated porcine IL3 still keeps the original stem cell mobilization function.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:
22154875
[PubMed - indexed for MEDLINE]
PMCID:
PMC3288236
Free PMC Article

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