Heterologous protein production using the Pichia pastoris expression system

Abstract

The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

“…P. pastoris is a well established heterologous expression system for the production of membrane proteins (45) and solubilized proteins were obtained from harvested P. pastoris membrane fragments in rather large amounts. To allow for functionality studies to be performed on those proteins, i.e.…”

Expression, Identification and Characterize of CD25-Binding Epitope Modified Human IL-2 in Pichia pastoris

“…However, degradation of target protein may occur during secretory expression especially in high-cell density culture [16]. Moreover, the degradation fragments often hinder effective purification of target protein [17]. In this experiment, the method of purification process was explored and finally the rhIL-2 was highly expressed by P. pastoris, a powerful eukaryotic expression system, reaching 1.35 g/L.…”

Expression of GFP using Pichia pastoris vectors with zeocin or G‐418 sulphate as the primary selectable marker

“…These issues are generally overcome by using alternative recombinant protein expression systems, such as P. pastoris (Macauley-Patrick et al, 2005;Sreekrishna et al, 1988;Eckart and Bussineau, 1996;Jahic et al, 2006;Hamilton and Gerngross, 2007). To date, P. pastoris has been successfully used for the expression of more than 420 heterologous recombinant proteins (MacauleyPatrick et al, 2005;Lin Cereghino et al, 2008;Boer et al, 2007).…”

“…At present, the methylotrophic yeast Pichia pastoris has been used for the production of over 420 heterologous proteins (Macauley-Patrick et al, 2005;Liu and Liu, 2008). It shares many of the attributes of yeasts such as Saccharomyces cerevisiae, including inter alia transformation with chromosomal integration vectors and gene replacement by homologous recombination (Cregg et al, 1985;Crane and Gould, 1994).…”