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Aspergillus
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Heterologous protein products will find application in the food industries and, increasingly so, in the pharmaceutical and chemicals industries. There is a paramount need to consider safety of the product and “food-acceptable” mold hosts have already proved to be the most widely investigated. Thus, the product should be free from contaminating material likely to be toxic, and the product itself should be authentic. Yield is also an important consideration, because it determines the success of a commercial operation, but, even in the research laboratory, yield is important because the methods used for structural studies of engineered enzymes, for example, generally require milligram quantities. These considerations determine the aspects discussed in the following, and some case studies will also be described.
Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Obtaining pure protein samples is a critical first step for many experiments. While a small number of highly abundant proteins can be purified directly from natural sources, most interesting proteins must be obtained by heterologous expressions or total chemical synthesis. The purification of a protein from a natural source requires large amounts of tissue containing the desired protein and extensive knowledge of the protein’s physicochemical properties or the use of immunoaffinity methodologies. As such, natural source isolation of the quantity of protein necessary for biochemical or biophysical studies is limited to a small number of proteins and will not be discussed here. Total chemical synthesis and expressed chemical ligation (ECL) technologies provide intriguing methods for the preparation of modified proteins, but these methods require techniques that are beyond the scope of many laboratories. A brief discussion of these techniques and their applications is provided at the end of the chapter. Generally, proteins are prepared by overexpression and purification using heterologous expression systems; as such, this will be the focus of this chapter. Heterologous expression has the advantage that it can be used for the preparation of small or large amounts of purified proteins, including modified and engineered proteins. Moreover, it can be used to express proteins from pathogenic organisms without the extensive biosafety precautions that would be required for handling the source organism and allows for the functional analysis of open reading frames.
Production of VNPs, VLPs, and Chimeras
Published in Nicole F Steinmetz, Marianne Manchester, Viral Nanoparticles, 2019
Nicole F Steinmetz, Marianne Manchester
Heterologous expression systems are of great interest as they allow the production of VLPs. VLPs are devoid of infectious nucleic acids and thus cannot replicate themselves. VLPs are considered safer from an agricultural point of view and human health perspective. Heterologous expression systems used to generate VLPs include use of bacteria, yeast, insect cells, and mammalian cells. All these systems have advantages and disadvantages in terms of yield, scale, time, costs, assembly efficiency, and biological integrity (reviewed in Schneemann & Young, 2003). In the following sections, we will discuss different systems and highlight their benefits and pitfalls (summarized in Table 3.1).
Production of bioactive recombinant monoclonal antibody fragment in periplasm of Escherichia coli expression system
Published in Preparative Biochemistry & Biotechnology, 2023
Preeti Saroha, Anurag S. Rathore
Amongst the broadly used expression system, E. coli is the most attractive choice of host for the heterologous proteins. However, due to high expression of recombinant proteins, many form insoluble aggregates within the cytoplasm, thereby resulting in formation of inclusion bodies. The protein in this form is devoid of any functional activity and hence, developing an efficient technique that can express the bioactive protein without compromising the expression level would lessen in the burden on downstream processing and also offer higher process yield. Although there is no universal approach that would be effective for all cases, numerous strategies have been attempted. Single promoter vectors are generally preferred for the expression of recombinant Fabs, with affinity tags for the ease of purification whereas recently upgraded vectors with dual promoters are slowly replacing the former ones due to expression of multi-domain heterologous proteins, such as antibody fragments, rendering a cost effective approach and simultaneously reducing the time of expression.[2,7] Plasmids are categorized based on the copy number as high copy number plasmids (>100) and low copy plasmids (10–12).[2,20]
Challenges of expressing recombinant human tissue factor as a secreted protein in Pichia pastoris
Published in Preparative Biochemistry & Biotechnology, 2022
Mohammad Jalili-Nik, Mohammad Soukhtanloo, Majid Mojarrad, Mohammad Hadi Sadeghian, Baratali Mashkani
Currently, P. pastoris has been suggested as a particularly efficient host for high-level expression of heterologous proteins based on the following advantages: the high growth rates, the capacity of providing high levels of the expressed protein, the comfort of applied techniques for genetic manipulation, the facility of performing different post-translational modifications and finally the feature of secreting low levels of endogenous proteins Karbalaei et al.,[9] Gasser et al.,[10] Cereghino and Cregg,[11] Zahrl et al.,[12] Juturu and Wu.[13] There are various signal sequences for foreign protein secretion in yeast, but the most widely used secretion signal for Pichia pastoris is the saccharomyces cerevisiae mating pheromone α- factor. The application of this secretory system is; however, not always promising and optimal secretion is highly dependent on the target protein. This study aimed to express TF in P. pastoris and evaluate the α-factor potency for secretion of three forms of TF, full-length TF (Full-TF), extracellular plus transmembrane domain (TED-TF) and only extracellular domain (ED-TF).
Standardized production of a homogeneous latex enzyme source overcoming seasonality and microenvironmental variables
Published in Preparative Biochemistry & Biotechnology, 2021
Sandro Rios Silveira, Raphael Alves Coelho, Brandon Ferraz e Sousa, Jefferson Soares de Oliveira, Laura Maria Isabel Lopez, José Vitor Moreira Lima-Filho, Pedro Abílio Vieira Rocha Júnior, Diego Pereira de Souza, Cleverson Diniz Teixeira de Freitas, Márcio Viana Ramos
Bioprospection, for new sources of enzymes, suitable for application in industrial processes is attracting increasing attention. In general, the use of enzymes, instead of chemicals, in industrial technologies is more environmentally friendly.[1] The challenge of prospecting for new enzymes is complex and laborious. The first challenges involve the choice of the biological source to be screened and possible systems for production. Heterologous expression of enzymes, in microorganisms, has been exploited. However, raw materials, obtained of agricultural practices to produce enzymes of market interest are still interesting. Due to the peculiar characteristics of enzymes and the particularities of the process associated to their use, many other aspects should be observed. It involves investigating the desired specificity and catalytic efficiency, pH and temperature requirements, suitability for large-scale use of the enzyme source and adequate storage conditions, among many other requirements.