title: Expression and purification of non-N-glycosylated porcine interleukin 3 in yeast Pichia pastoris.
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.
authors.affiliation: Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: Christina E
authors.initials: CE
authors.lastname: Hermanrud
authors.forename: Vimukthi
authors.initials: V
authors.lastname: Pathiraja
authors.forename: Abraham
authors.initials: A
authors.lastname: Matar
authors.forename: Raimon
authors.initials: R
authors.lastname: Duran-Struuck
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Crepeau
authors.forename: Srimathi
authors.initials: S
authors.lastname: Srinivasan
authors.forename: David H
authors.initials: DH
authors.lastname: Sachs
authors.forename: Christene A
authors.initials: CA
authors.lastname: Huang
authors.forename: Zhirui
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015870:Gene Expression; D006031:Glycosylation; D007377:Interleukin-3 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013552:Swine / Q000235:genetics / Q000276:immunology*
chemical_list: D007377:Interleukin-3; D011994:Recombinant Proteins