Expression and characterization of human glycosylated interleukin-1 receptor antagonist in Pichia pastoris.
Interleukin-1 receptor antagonist is an inhibitor of the pro-inflammatory action of interleukin-1. The gene encoding for interleukin-1 receptor antagonist (IL-1ra) was cloned into a Pichia pastoris expression vector pPICzalphaA (Invitrogen, USA) and transformed into P. pastoris strain SMD1168H. Multi-copy selection of the gene produced a high expressing strain of IL-1ra that produced 17mg/L of total secreted purified protein. The IL-1ra produced in P. pastoris was a mixture of glycosylated and non-glycosylated IL-1ra where 70% of the total protein was glycosylated. SP-Sepharose purification allowed for separation of the two expressed forms of IL-1ra, which permits biochemical investigation of glycosylated and non-glycosylated IL-1ra using one expression system. Mass spectrometric analysis revealed the expression of the full-length protein and that the glycosylated IL-1ra contained high mannose glycoforms that ranged from Man(9)GlcNAc(2) to Man(14)GlcNAc(2).
Hamilton
Brian S
BS
Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, IN 47401, USA.
Brede
Yvonne
Y
Tolbert
Thomas J
TJ
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008358:Mannose / Q000032:analysis; D013058:Mass Spectrometry; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D053590:Interleukin 1 Receptor Antagonist Protein; D011994:Recombinant Proteins; D008358:Mannose