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Journal of Cellular Physiology Sep 2020One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression... (Review)
Review
One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein.
Topics: Fungal Proteins; Proprotein Convertases; Protein Biosynthesis; Proteomics; Recombinant Proteins; Saccharomyces cerevisiae Proteins; Saccharomycetales
PubMed: 32057111
DOI: 10.1002/jcp.29583 -
Current Protocols in Protein Science Aug 2010Recombinant protein expression in Escherichia coli (E. coli) is simple, fast, inexpensive, and robust, with the expressed protein comprising up to 50 percent of the... (Review)
Review
Recombinant protein expression in Escherichia coli (E. coli) is simple, fast, inexpensive, and robust, with the expressed protein comprising up to 50 percent of the total cellular protein. However, it also has disadvantages. For example, the rapidity of bacterial protein expression often results in unfolded/misfolded proteins, especially for heterologous proteins that require longer times and/or molecular chaperones to fold correctly. In addition, the highly reductive environment of the bacterial cytosol and the inability of E. coli to perform several eukaryotic post-translational modifications results in the insoluble expression of proteins that require these modifications for folding and activity. Fortunately, multiple, novel reagents and techniques have been developed that allow for the efficient, soluble production of a diverse range of heterologous proteins in E. coli. This overview describes variables at each stage of a protein expression experiment that can influence solubility and offers a summary of strategies used to optimize soluble expression in E. coli.
Topics: Escherichia coli; Protein Engineering; Protein Folding; Proteomics; Recombinant Proteins; Solubility
PubMed: 20814932
DOI: 10.1002/0471140864.ps0524s61 -
Protein Science : a Publication of the... Aug 2019The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein... (Review)
Review
The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost-effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.
Topics: Escherichia coli; Recombinant Proteins
PubMed: 31219641
DOI: 10.1002/pro.3668 -
Applied Microbiology and Biotechnology Feb 2023Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant... (Review)
Review
Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed. KEY POINTS: • Fed-batch culture in CHO cells is reviewed. • Fed-batch has become a common technology for recombinant protein production. • Fed batch culture promotes recombinant protein production in CHO cells.
Topics: Cricetinae; Animals; Humans; Batch Cell Culture Techniques; Cricetulus; CHO Cells; Recombinant Proteins; Bioreactors; Immunoglobulins
PubMed: 36648523
DOI: 10.1007/s00253-022-12342-x -
Current Protocols in Protein Science Apr 2015When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and... (Review)
Review
When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same.
Topics: Affinity Labels; Chromatography, Affinity; Escherichia coli; Recombinant Proteins
PubMed: 25829302
DOI: 10.1002/0471140864.ps0601s80 -
Open Biology Aug 2016The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for... (Review)
Review
The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design.
Topics: Cloning, Molecular; Escherichia coli; Gene Expression; Protein Engineering; Protein Processing, Post-Translational; RNA Folding; RNA, Messenger; Recombinant Proteins
PubMed: 27581654
DOI: 10.1098/rsob.160196 -
Postepy Higieny I Medycyny... Mar 2013For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the... (Review)
Review
For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities.
Topics: Bacillus; Bacteria; Escherichia coli; Gene Expression; Genetic Vectors; Recombinant Proteins
PubMed: 23475488
DOI: 10.5604/17322693.1038351 -
Microbial Cell Factories Mar 2015Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from... (Review)
Review
Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.
Topics: Escherichia coli; Inclusion Bodies; Models, Molecular; Protein Denaturation; Protein Folding; Protein Refolding; Protein Unfolding; Recombinant Proteins; Solubility
PubMed: 25889252
DOI: 10.1186/s12934-015-0222-8 -
Cells Jul 2021The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to... (Review)
Review
The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line's most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering.
Topics: CRISPR-Cas Systems; Cell Adhesion; Cell Engineering; Cell Proliferation; Clone Cells; Gene Editing; Gene Expression Regulation; Genetic Vectors; HEK293 Cells; Humans; Recombinant Proteins; Transfection; Transgenes
PubMed: 34359846
DOI: 10.3390/cells10071667 -
Applied Microbiology and Biotechnology Jun 2014Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a... (Review)
Review
Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.
Topics: Gene Expression; Industrial Microbiology; Pichia; Recombinant Proteins
PubMed: 24743983
DOI: 10.1007/s00253-014-5732-5