Open Access Medical Books

TEXTBOOK : PROTEIN ENGINEERING - TECHNOLOGY AND APPLICATION

Edited by Tomohisa Ogawa .

Open Access .
196 pages .

Yeasts are the favorite alternative hosts for the expression of heterologous proteins for research, industrial or medical use [1]. As unicellulars microorganism have the advantages of bacteria as ease of manipulation and growth rate. But comparing with bacterial system, they are capable of many of the post-translational modifications performed by higher eukaryotic cells, such as proteolytic processing, folding, disulfide bond formation and glycosylation [2].
Historically Saccharomyces cerevisiae has been the most used yeast host due to the large amount of knowledge on genetics, molecular biology and physiology accumulated for this microorganism [3-5]. However, it was rapidly found to have certain limitations: low product yields, poor plasmid stability, hyperglycosylation and low secretion capacities. These limitations are now relieved by a battery of alternative yeast as cell factories to produce recombinant proteins.
Some of these alternative yeast cell factories are fission yeast as Schizosaccharomyces pombe [6], Kluyveromyces lactis [7], methylotrophic species as Pichia pastoris [8], Candida boidinii [9], Pichia methanolica [10], Hansenula polymorpha [11], and the dimorphic species Yarrowia lipolytica [12], and Arxula adeninivorans [13]. It is very usual that the performance of these alternative hosts frequently surpass those of S. cerevisiae in terms of product yield, reduced hyperglycosylation and secretion efficiency, especially for high molecular weight proteins [14].
Several reviews compare advantages and limitations of expression systems for foreign genes [15-20]. Between them Pichia pastoris has emerged in the last decade as the favorite yeast cell factory for the production of heterologous proteins. A search in ISI Web of knowledge (web of science) with the keywords microorganism+ heterologous protein P.
pastoris is the preferred host (667 entrances) followed by Candida and Schizosaccharomyces (161 and 124 entrances respectively). Specifically for heterologous lipase production P. pastoris is the most used host [21].
Why P. pastoris emerged as an excellent host system to produce recombinant products?. The story started one decade after oil crisis in the 70’s when Phillips Petroleum and the Salk Institute Biotechnology/Industrial Associates Inc. (SIBIA, La Jolla, Ca, USA) used Pichia as a host system for heterologous protein expression [22-24]. Nowadays, more than 500 proteins have been expressed using this system [25] and it also has been selected by several protein production platforms for structural genomics programs [26]. P. pastoris combines the ability of growing on minimal medium at very high cell densities (higher than 100 g DCW/L), secreting the heterologous protein simplifying their recovery. Also, it performs many of the higher eukaryotic post-translational modifications such as protein folding, proteolytic processing, disulfide bond and glycosylation [24]. However, it has been shown that both, N- and O-linked oligosaccharide structures, are quite different from mammalian cells, for example, they are of a heterogeneous high-mannose type. The consequence is that high mannose type N-glycans attached to recombinant glycoproteins can be cleared rapidly from the human bloodstream, and they can cause immunogenic reactions in humans [27]. Nevertheless GlycoFi’s glycoengineering technology allows the generation of yeast stains capable of replicating the most essential steps of the N-glycosylation pathway found in mammals [28]. But, probably the most important characteristic of P. pastoris is the existence of a strong and tightly regulated promoter from alcohol oxidase 1 gene, PAOX1. Thus, methanol was used as carbon source and inducer of heterologous protein production in this system [29].
Daly and Hearn [30] reviewed various aspects of the P. pastoris expression system and also consider the factors that need to be taken into account to achieve successful recombinant protein expression, particular when more complex systems are contemplated, such as those used in tandem gene or multiple gene copy experiments. Between them, several genetic and physiological factors such as the codon usage of the expression gene, the gene copy number, efficient transcription by using strong promoters, translation signals, translocation determined by the secretion signal peptide, processing and folding in the endoplasmatic reticulum and Golgy and, finally, secretion out of the cell, as well as protein turnovers by proteolysis, but also of the optimization of fermentation strategy [31].
The objective of this chapter is to review the classic and alternative operational strategies to maximize yield and/or productivity from an industrial point of view and also how to obtain a repetitive product from batch to batch applying process analytical technology (BioPAT) .....

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CONTENTS : 

Section 1 of the Textbook : Basic Technology of Protein Engineering .


 1 Bioprocess Engineering of Pichia pastoris, an Exciting Host Eukaryotic Cell Expression System 3 Francisco Valero

 2 Chromatography Method 33 Jingjing Li, Wei Han and Yan Yu

 3 Protein-Protein and Protein-Ligand Docking 63 Alejandra Hernández-Santoyo, Aldo Yair Tenorio-Barajas, Victor Altuzar, Héctor Vivanco-Cid and Claudia Mendoza-Barrera

Section 2 of the Textbook : Application of Protein Engineering .


 4 Applications of the In Vitro Virus (IVV) Method for Various Protein Functional Analyses 85 Noriko Tabata, Kenichi Horisawa and Hiroshi Yanagawa

 5 Experimental Molecular Archeology: Reconstruction of Ancestral Mutants and Evolutionary History of Proteins as a New Approach in Protein Engineering 111 Tomohisa Ogawa and Tsuyoshi Shirai

 6 Protein Engineering of Enzymes Involved in Bioplastic Metabolism 133 Tomohiro Hiraishi and Seiichi Taguchi

 7 Identification of HMGB1-Binding Components Using Affinity Column Chromatography 167 Ari Rouhiainen, Helena Tukiainen, Pia Siljander and Heikki Rauvala




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