Moulton Fed-Batch Fermentation

A Practical Guide to Scalable Recombinant Protein Production in Escherichia Coli
1. Auflage 2014
ISBN: 978-1-908818-33-1
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 188 Seiten

Reihe: Woodhead Publishing Series in Biomedicine

Fed-batch Fermentation is primarily a practical guide for recombinant protein production in E. coli using a Fed-batch Fermentation process. Ideal users of this guide are teaching labs and R&D labs that need a quick and reproducible process for recombinant protein production. It may also be used as a template for the production of recombinant protein product for use in clinical trials. The guide highlights a method whereby a medium cell density - final Ods = 30-40 (A600) - Fed-batch Fermentation process can be accomplished within a single day with minimal supervision. This process can also be done on a small (2L) scale that is scalable to 30L or more. All reagents (media, carbon source, plasmid vector and host cell) used are widely available and are relatively inexpensive. This method has been used to produce three different protein products following cGMP guidelines for Phase I clinical studies. - This process can be used as a teaching tool for the inexperienced fermentation student or researcher in the fields of bioprocessing and bioreactors. It is an important segue from E. coli shake flask cultures to bioreactor - The fed-batch fermentation is designed to be accomplished in a single day with the preparation work being done on the day prior - The fed-batch fermentation described in this book is a robust process and can be easily scaled for CMO production of protein product

Garner G. Moulton has a Masters of Science in Biology from the Program of Cell and Molecular Biology at San Diego State University. He has been working in biotech and non-profit research for over 25 years, and has focussed specifically on protein purification and fermentation process development over the last 17 years. During the past 4 years he has been employed by the Infectious Disease Research Institute (IDRI) in Seattle, WA, USA, where he is a principal research associate in charge of the Process Development Department. He has developed many robust fermentation processes that can be adapted to many different recombinant products without having to re-optimize conditions of growth and expression. Numerous projects have been successfully transferred to a contract manufacturer for scale-up production and use in clinical trials.

Autoren/Hrsg.

Moulton, G G

Leseproben

2 Generation of a recombinant Escherichia coli expression system
Abstract
When generating a recombinant cell line that expresses a foreign gene of interest, the work starts with an E. coli host strain such as HMS-174 (DE3) or BL21 (DE3), a plasmid of choice such as pET-28a or pET-29a, and a gene of interest (usually a protein that is needed for diagnostic or therapeutic use). A sufficient expression screening process of different host cells should be performed, which allows expression analysis of the protein of interest in terms of expression fidelity, protein quality and stability. This process matches the host cell machinery (protease profile, protein trafficking, etc.) with the specific properties of the recombinant protein product (stability, hydrophobic character, toxicity to host cell, etc.). This chapter presents a short review of the transcription and translation processes that occur within the recombinant cell. Key words plasmid host cell bacteria recombinant E. coli cloning transcription translation expression gene protein competent media 2XYS antibiotic selection working cell bank transformation 2.1 Plasmids
As was stated in the introduction, E. coli was the first microorganism to be thoroughly analyzed by both genetic and molecular biological means. This led to it also being the first to be used for genetic engineering and recombinant protein production. Even though much is known about the bacterium E. coli, it is certainly not a trivial task to set up a recombinant system within this host, or any host for that matter. Some of the main issues to address in a recombinant E. coli system are the instability of the plasmid vectors, initiation and translation problems, along with mRNA stability. Plasmids are generally small circular DNA structures found in many different strains of bacteria. They can also be found in large numbers, over 100 copies per cell, and are self-replicating. In recombinant bacteria such as E. coli, this allows for the production of significant amounts of plasmid DNA or recombinant proteins (potentially) from a small volume of cells. For the purposes of recombinant DNA or protein production, plasmids have been developed with a number of advantages for cloning. Almost all plasmids carry a gene or genes that encode for antibiotic resistance. This antibiotic selection allows the host cell to grow in the media in the presence of an antibiotic such as kanamycin. If a cell looses the plasmid or the antibiotic resistant gene, the cell is killed. This helps ensure the continued presence of the plasmid as the cell culture grows and is ultimately harvested for the plasmid DNA or an induced foreign protein product. This is important in terms of recombinant E. coli high cell density fermentations when the cells go through many divisions. Without the antibiotic selection, the culture would eventually contain a heterogeneous population of cells, some with plasmid and some without. The plasmid-free cells would not have the metabolic pressure of maintaining the plasmid copies and thus would grow at a faster rate, eventually outgrowing the plasmid containing cells and reducing the recombinant production fidelity of the culture. One of the most important recombinant elements of the plasmid is the promoter used to initiate expression of a recombinant protein of interest. Promoters are areas within the plasmid that are responsible for the enlistment and binding of the RNA polymerase (RNAP) to different transcription subunits. These transcription factors play a significant role in the control of transcription within the E. coli. The transcription factors are proteins that bind to specific DNA sequences and thus control the rate of transcription of the DNA sequence to the messenger RNA (mRNA) sequence. These factors can act to either inhibit transcription (repress) or stimulate transcription (activate) by binding to or effecting the binding of the RNAP to the DNA. Therefore, transcription is regulated by more than one transcription factor in most bacterial promoters, which allows for tighter control and the ability to respond to different environmental growth conditions when needed. The discovery of the ColE1 plasmid [66] led to the construction of the first plasmid based vectors in biotechnology, and were found to be essential tools for the cloning and production of recombinant proteins in E. coli. The pUC18 plasmid is one example of an early vector used for recombinant protein production. Expression vectors, or plasmids, contain an origin of replication, an antibiotic resistant marker and an expression cassette that regulates the recombinant gene transcription and translation (Figure 2.1). Many plasmid vectors in use today contain phage polymerase promoters such as the T7 polymerase/promoter. Some will contain two distinct promoters, which are inserted on each side of the cloning site. This allows for transcription of either strand of the recombinant DNA sequence. As shown in Figure 2.1, the pET system contains the promoter for the gene encoding for the T7 RNAP. The T7 RNAP is usually supplied by the recombinant bacterial cell in the form of a ? lysogen, which expresses the polymerase gene under control of the lacUV5 promoter [67]. Figure 2.1 Generic plasmid. The gene of interest will be inserted between two restriction sites that contains a T7 promoter in frame with and adjacent to the gene of interest Other characteristics of the expression vectors that are used include plasmid stability and DNA structure or gene structure for transfer to other bacterial hosts. There has been some concern that when using a strong promoter such as the T7, coupled with a large plasmid copy number (>150 copies per cell), the cell machinery becomes overwhelmed and causes a disruption of the recombinant protein synthesis if not all protein synthesis within the cell. This was commonly seen with the pUC recombinant vectors. A new vector (ColE1-type) has been developed to address this issue, successfully controlling/maintaining the copy number during strong induction conditions [68,69]. Unfortunately, the ColE1-type vectors tend to be unstable when grown at high cell densities. As mentioned above, this is due to the large number of divisions taking place and the fact that the host strain is recA+. With a high copy number of plasmids in recA+ strains, the plasmids have an increasing chance of homologous recombination, which converts them to head-tail dimeric plasmids. Given that these dimeric plasmids have two ori sequences instead of one they will, theoretically, replicate twice as fast as the single plasmids, eventually overtaking the single plasmid population and causing copy number depression by interfering with cellular circuitry [70]. This problem is clearly observed in the recombinant strain W3110, which is recA+. At the end of fed-batch fermentation without antibiotic selection, 50% of the cells had lost their plasmids. In recA mutated strains such as HMS-174/DE3 (recA1), this is not a problem and has been used successfully by the author in fed-batch fermentations without antibiotic selection. The promoters of native plasmids in bacteria are usually part of operons that control the catabolism of carbohydrates, both negatively and positively. One of the most well-known and used promoters is the E. coli lac operon, which is controlled by the presence or absence of lactose. In the absence of lactose, the LacI repressor binds to a region downstream of the lac promoter (region +1 to +21) prohibiting the transcription of lac gene series, lacZYA. The operon consists of three structural genes in series, lacZ, lacY and lacA. In the presence of allolactose, which is made from lactose, this inhibition is reversed. The allolactose binds to the repressor, which in turn releases it from the lac promoter and allows the transcription of the lacZYA genes to be turned on. In the case of a recombinant system where the cell is being asked to produce a recombinant protein, this operon is turned on by the addition of the lactose mimetic, Isopropyl ß-D-1-thiogalactopyranoside (IPTG), to the growing E. coli culture. Complications can arise when using negatively regulated promoters and growing the recombinant bacterial culture to a high cell density. In the bacterial cell, the LacI gene only translates a tiny amount of repressor molecules compared to the presence of the number of operons that are needed to remain repressed, as is the case with high copy number plasmids. This high copy number of plasmids, when induced, puts a large amount of metabolic pressure on the host cell, slowing its growth and metabolism. The recombinant protein DNA is recombined into a plasmid cloning site so that the transcription will be driven by the T7 RNAP associated with the lac operon. This polymerase is, in turn, controlled (regulated) by the presence or absence of the lactose mimetic, IPTG, which is added to the medium during induction of the recombinant protein product. The lac operon is used in bacteria such as E. coli to support the transport and...