Optimization of the expression of recombinant human activin A in the yeast Pichia pastoris

Abstract

We report a new procedure to express recombinant human activin A using the methanolic yeast, Pichia pastoris. Optimization of culture procedures has involved comprehensive examination of the effects of culture vessel shape, volume of broth in the induction and expression cultures, methanol concentration, culturing temperature, and pH of the expression cultures. After this optimization, as well as modification of the native cleavage sites, a laboratory scale procedure has been established which routinely produced 2-10 mg/L amounts of this vital growth factor in the highly efficient, eukaryotic yeast system. This system avoids the need to produce this protein and similar TGF-beta proteins in mammalian cell lines which, in addition to being costly, produce many native binding partners of these cystine knot proteins, a factor which can dramatically affect yields of the target protein.

Production, Isolation, and Structural Analysis of Ligands and Receptors of the TGF-β Superfamily

“…There are several reports of the overexpression of mammalian signaling proteins in non-mammalian cells, including human BMP-2, bovine activin A, and bovine inhibin A in insect cells [42,43] and human activin A in yeast [44]. These expression hosts have some advantages over mammalian cells, including the absence of native binding partners that can interfere with maturation [18] and potentially higher yields [44,45]. The higher yields have only been obtained when the protease cleavage site between the pro-domain and mature signaling dimer have been altered to more closely match the endogenous proteases in the expression host [44,45].…”

“…These expression hosts have some advantages over mammalian cells, including the absence of native binding partners that can interfere with maturation [18] and potentially higher yields [44,45]. The higher yields have only been obtained when the protease cleavage site between the pro-domain and mature signaling dimer have been altered to more closely match the endogenous proteases in the expression host [44,45]. Thus, successful heterologous expression has required some alterations to account for differences in molecular machinery relative to mammalian hosts, but so far these have been relegated to the enzymes responsible for proteolytic processing, not enzymes involved in catalyzing disulfide exchange or folding.…”

Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastorisan alpha 1-antitrypsin in Pichia pastoris

“…In the simplest method, one can optimize conditions of the culture such as culture medium, cell density, inducer concentration, temperature, pH, aeration, etc. (Maldonado et al 2007;Maldonado et al 2008;Zhao et al 2008;Fredericks et al 2010;Varghese et al 2010). However, most of the time, improving external factors is not sufficient, whereas changing some genetic factors can significantly impact the production of recombinant protein Li et al 2008).…”

Production of the virus‐like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents

“…The recombinant plasmid, pPICZ B‐M f (10 μg) was linearized using Sac I and introduced into P. pastoris X33 strain via electroporation (Micropulser, BioRad, Hercules, CA) at 2 kV for ∼5 ms as described by Fredericks et al After transformation, the yeast cells were plated on the Yeast Extract Peptone Dextrose agar containing zeocin (100 μg/mL; Invitrogen, USA). In order to select for clones with higher resistance level, transformants were plated on Yeast Extract Peptone Dextrose agar containing increasing concentration of zeocin (250, 500, and 1,000 μg/mL).…”