Playing catch-up with escherichia coli: Using yeast to increase success rates in recombinant protein production experiments (original) (raw)
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Optimising yeast as a host for recombinant protein production (review)
2012
Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesising a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, whilst also being quick, easy and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production.
Comparison of Yeasts as Hosts for Recombinant Protein Production
Microorganisms, 2018
Recombinant protein production emerged in the early 1980s with the development of genetic engineering tools, which represented a compelling alternative to protein extraction from natural sources. Over the years, a high level of heterologous protein was made possible in a variety of hosts ranging from the bacteria Escherichia coli to mammalian cells. Recombinant protein importance is represented by its market size, which reached 1654millionin2016andisexpectedtoreach1654 million in 2016 and is expected to reach 1654millionin2016andisexpectedtoreach2850.5 million by 2022. Among the available hosts, yeasts have been used for producing a great variety of proteins applied to chemicals, fuels, food, and pharmaceuticals, being one of the most used hosts for recombinant production nowadays. Historically, Saccharomyces cerevisiae was the dominant yeast host for heterologous protein production. Lately, other yeasts such as Komagataella sp., Kluyveromyces lactis, and Yarrowia lipolytica have emerged as advantageous hosts. In this review, a comparative analysis is done listing the advantages and disadvantages of using each host regarding the availability of genetic tools, strategies for cultivation in bioreactors, and the main techniques utilized for protein purification. Finally, examples of each host will be discussed regarding the total amount of protein recovered and its bioactivity due to correct folding and glycosylation patterns.
Annals of the New York Academy of Sciences, 1994
The yeast Saccharomyces cerevisiae has proven to be an excellent host for the production of a number of different recombinant proteins that have potential medical and commercial applications. The use of S. cerevisiae as a recombinant host has a number of advantages: (1) yeast cells are easily fermented to industrial scale using simple media; (2) yeast cells are free of endotoxin and nonpathogenic to man; (3) S. cerevisiae has well-developed genetics, which offers unparalleled possibilities for solving problems that may exist at various steps in the production of heterologous proteins through a combination of classical and molecular genetic approaches; and (4) yeast cells are capable of performing post-translational and cotranslational processing of proteins in a manner similar to higher eukaryotes. In addition, the secretion of heterologous proteins by yeast has several advantages: first, only low levels of native proteins are secreted into the culture medium, simplifying purification of a target protein; second, yeast is able in many cases to correctly fold proteins and form intramolecular disulfide bonds during secretion as demonstrated by the successful secretion of proteins containing multiple disulfide bonds in a biologically active f~r m. I-~ Two examples are echistatin4 and anti~tasin.~ Finally, S. cerevisiae has a number of strong promoters that are either inducible or constitutive. These promoters have been used in a variety of different yeast expression vectors that in turn can be used to readily transform yeast using several different selective markers (URA3, LEU2, T W I , etc.). As many of the above features of heterologous protein expression in yeast have been discussed in several recent they will not be discussed further here. This paper will highlight the well-defined genetics of S. cerevisiae, which enable one to engineer yeast host strains with desired genetic characteristics such that
Understanding the yeast host cell response to recombinant membrane protein production
Biochemical Society transactions, 2011
Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membr...
Yeast synthetic biology for the production of recombinant therapeutic proteins
FEMS Yeast Research, 2014
The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins.
Recombinant Protein Production in Yeasts
Molecular Biotechnology, 2005
Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. These technologies, now approx 25 yr old, have become one of the most important technologies developed in the twentieth century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances in rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we summarize advantages and limitations of the main and most promising yeast hosts.
Yeasts become the most preferred expression system for the production of recombinant proteins which play an important role in the development of biopharmaceutical products, antibodies for disease treatment, and enzymes for the food industries. The ability to grow in simple media, and ease of genetic manipulation with the benefits of typical eukaryotic expression which include protein processing, folding, and post-translational modifications, have pushed them as one of the emerging hosts for recombinant protein production. Furthermore, yeasts are additionally quicker, easy to use, and cost-effective with high yield production in comparison to higher expression hosts. The effective productivity of the recombinant proteins is also influenced by the external parameters. This paper reviews different optimization methods of the recombinant protein production for several factors such as pH, temperature, media, agitation rate, inducer, inoculum size and induction time using one factor at a time (OFAT), Response Surface Methodology (RSM) and Artificial Neural Network (ANN). This review highlights the current studies regarding the optimization of the recombinant proteins expressed in three different yeasts namely; Saccharomyces cerevisiae, Komagataella phaffii, and Yarrowia lipolytica. These are the critical parameters which can be used to optimize the recombinant protein in yeast systems. The purification methods used to purify the proteins are also discussed for each system.
Production of recombinant proteins and metabolites in yeasts
Applied Microbiology and Biotechnology, 2011
Recombinant DNA (rDNA) technologies allow the production of a wide range of peptides, proteins and metabolites from naturally non-producing cells. Since human insulin was the first heterologous compound produced in a laboratory in 1977, rDNA technology has become one of the most important technologies developed in the 20th century. Recombinant protein and metabolites production is a multi-billion dollar market. The development of a new product begins with the choice of the cell factory. The final application of the compound dictates the main criteria that should be taken into consideration: (1) quality, (2) quantity, (3) yield and (4) space time yield of the desired product. Quantity and quality are the most predominant requirements that must be considered for the commercial production of a protein. Quantity and yield are the requirements for the production of a metabolite. Finally, space time yield is crucial for any production process. It therefore becomes clear why the perfect host does not exist yet, and why-despite important advances in rDNA applications in higher eukaryotic cells-microbial biodiversity continues to represent a potential source of attractive cell factories. In this review, we compare the advantages and limitations of the principal yeast and bacterial workhorse systems.
Recombinant Protein Production in Yeast
Methods in molecular biology, 2012
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