title: Expression of the human activin type I and II receptor extracellular domains in Pichia pastoris.
abstract: Methods for the expression in Pichia pastoris and purification of the human activin receptor type I and II extracellular domains (ARIa/ARIb-ECDs, ARIIA/ARIIB-ECDs) are described. Key experimental aspects are also documented of the vector transformation methodology and the binding characteristics of these ECDs with activin A and inhibin. The cDNA constructs for these ECDs contained a C-terminal His6-tag with either the native signal (N) or the yeast alpha mating factor (alphaMF) sequence and were introduced into the pPICZ expression vector either as a single-copy or as a four-copy expression cassette. Hyper-resistant transformants (zeo(R): 500 microg/mL) generated from the cassette containing a single copy of the expression vector gave the stronger signal intensity with a DNA dot-blot screening assay. These transformants also produced higher quantities of the corresponding recombinant protein compared to transformants using the four-copy cassette vector. All receptor-ECD proteins expressed were found to be heterogeneously glycosylated, whereby the ARIIA-ECD and ARIIB-ECD had undergone two Asn-linked glycosylation events and the ARIb-ECD a single event. By SDS-PAGE, the de-glycosylated proteins migrated larger than the expected core size, indicating that they may have undergone O-linked glycosylation. Biacore-based procedures with the glycosylated and de-glycosylated ARIIA-ECD were employed to determine the kinetic and equilibrium binding parameters for the interaction with activin A and inhibin. The glycosylated ARIIA-ECD bound to activin A with a KD of 11.9 nM and inhibin with a KD of 21.1 nM. Although glycosylation of ARIIA-ECD was not strictly required for high affinity interactions with activin A or inhibin, it markedly improved the overall stability of the ARIIA-ECD.
authors.affiliation: ARC Special Research Centre for Green Chemistry, Monash University, Wellington Road, Clayton, Vic. 3800, Australia.
authors.forename: Rachel
authors.initials: R
authors.lastname: Daly
authors.forename: Milton T W
authors.initials: MT
authors.lastname: Hearn
mesh_terms: D030201:Activin Receptors, Type I / Q000096:biosynthesis* / Q000235:genetics; D030301:Activin Receptors, Type II / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D010843:Pichia; D046188:Protein Modification, Translational / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D011993:Recombinant Fusion Proteins; D030201:Activin Receptors, Type I; D030301:Activin Receptors, Type II; C425585:activin receptor type II-A; C425584:activin receptor type II-B