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Gene Therapy for Acquired Tissue Damage
Published in Yashwant V. Pathak, Gene Delivery Systems, 2022
Rakesh Sharma, Robert Moffatt, Yuvraj Singh Negi, Shashi Prabha Singh
Bacterial cell banks should be tested for phenotypic and genomic identity. The presence or absence of inserted or deleted sequences necessary for the safe use of the GTMP should be confirmed. The immunological identity, including the genetically modified components, should be determined, for instance, by serotyping. Absence of contaminating bacteria, contaminating plasmids and contaminating bacteriophage particles that can infect the bacterial producer strain, fungal sterility, and intervial homogeneity of cell bank stocks should be assured. For transformed bacterial cell banks, testing should include the presence of plasmid or genome sequences containing the therapeutic sequence and associated regulatory/control elements, plasmid copy number and ratio of cells with/without plasmids. The principle is described in ICH guideline Q5D on derivation and characterization of cell substrates.
Escherichia coli
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Hisashi Yasueda, Hiroshi Matsui
A high-copy-number plasmid vector is considered to be useful for ensuring the expression of a desired gene because of the gene dosage effect. However, the constitutive overproduction of potentially toxic proteins by the elevated gene dosage is usually detrimental to host cell growth. One solution to this problem is to use runaway replication plasmid vectors. The first runaway plasmid vectors, Rl-derived plasmids, were described by Uhlin et al. [81]. By using these vectors, the expression of recA [82], T4 DNA ligase [83], bovine growth hormone (bGH) [49], and human superoxide dismutase (hSOD) [84] has been reported. To avoid the problem of derepression that may arise when using a high-copy-number plasmid, Chew et al. have developed a genetic system that simultaneously controls the replication and expression of cloned genes [85]. Their principle was to fuse a pBR322-on immediately downstream from the target gene so that the primer for replication would be cotranscribed with the gene. Thus, upon the induction of transcription, both the plasmid copy number and the amount of transcripts were increased. Another attempt to increase the gene dosage involves the construction of tandem or multiple gene structures in plasmids. These multiple expression units are composed of a promoter, RBS, foreign protein-encoding region, and transcription terminator signal.
The Needs for Sensors in Bacterial and Yeast Fermentations
Published in John V. Twork, Alexander M. Yacynych, Sensors in Bioprocess Control, 2020
Bruce F. Bishop, Stephen J. Lorbert
As elegant as the foregoing strategies appear to be, they are finding limited application in the baker’s yeast industry. In a recent publication, Chen and Chiger state, “In practice, baker’s yeast is not propagated with strictly exponential substrate additions, nor is it propagated at the highest specific growth rate compatible with maximal substrate yield. Rather, baker’s yeast propagation is conducted to produce specific properties in the final yeast product” [43]. The commercial baker’s yeast fermentation process attempts to maximize those characteristics in the yeast which give the greatest utility to the end product. Parameters such as leavening activity, appearance, and storage stability are not necessarily optimized under the same conditions that result in maximum biomass production. This is also true of recombinant yeast and bacterial fermentation systems where the conditions for product production are not necessarily the same as those which maximize the production of biomass. For example, in recombinant bacterial systems, a temperature shift can be employed for some types of vector systems to increase the number of plasmid copies inside the cells. By increasing the average copy number, it is possible to amplify the expression of the recombinant protein [30,44]. Typically, the bacteria are initially grown at a temperature that is optimal for growth. When an appropriate cell density is reached in the fermentation, the temperature is raised to cause an increase in the plasmid copy number. Ideally, this results in the expression of large amounts of the recombinant protein in conjunction with a relatively low-level constitutive promoter. In a sense, two fermentations must be optimized: one for growth and one for product expression. A good carbon source feeding strategy is a primary factor influencing the productivity of both phases of the fermentation. Feeding strategies which minimize ethanol and organic acid production are likely to have immediate benefits in yeast and bacterial fermentation processes that are oriented toward the production of recombinant proteins. From that viewpoint, the control strategies described earlier for baker’s yeast may be readily applicable to yeast fermentation processes that are directed toward the production of recombinant proteins. At this point, however, there is no published information describing the use of such strategies for yeast.
Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: insights from structure-function relationship for novel applications
Published in Preparative Biochemistry & Biotechnology, 2023
Kajal Kachhawaha, Santanu Singh, Khyati Joshi, Priyanka Nain, Sumit K. Singh
In addition to the codon bias, low expression of proteins could be caused by protein toxicity either before or after induction.[15] For the former, controlling basal induction by tightly regulated promoters enhances the protein expression multi-fold. Also, for expression vectors containing lac-based promoters, the use of defined media with glucose as the primary carbon source is preferred.[10] Similarly, for T7-based systems, using pLysS/pLysE bearing E. coli BL21(DE3) cells help in reducing protein toxicity by controlling the expression levels of T7 RNA polymerase.[13] On the contrary, for the cases of reduced expression due to protein toxicity after induction, using tunable promoters and reducing induction time are some of the reported approaches to enhance protein expression.[58] In addition, directing the protein expression to the E. coli periplasm and using a lower plasmid copy number during recombinant protein expression allows for utilizing the oxidative environment of the former and reducing the metabolic burden on the cells owing to the latter.[20] Further, increasing biomass by optimizing and using novel media additives and providing appropriate culture conditions (aeration and anti-foaming) also boosts the recombinant protein expression.[64]
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
Gene synthesis was done by Synbio Technologies. Codon optimized gene of TNFα inhibitor antibody fragment was cloned into a dual promotor high plasmid copy number vector pRSF-Duet vector, with outer membrane protein A (Omp A) signal sequence upstream of both light and heavy chain in MCS1 and MCS2 under the regulation of T7 promotor, respectively. Accuracy of the sequence was confirmed using DNA sequencing and PCR. One microliter of DNA was transformed into E. coli (BL21 DE3) competent cells, heat shocked at 42 °C for 45 s, and plated on LB agar media. Colonies were selected based on colony PCR using T7 universal primers. Conditions used for PCR are as follows: Initial denaturation at 95 °C (2 min), followed by 30 cycles of denaturation (95 °C) for 60 s, annealing (55 °C) for 45 s, and extension (72 °C) for 60 s and final extension (72 °C) for 10 min followed by termination of reaction at 4 °C.
Optimization of the 503 antigen induction strategy of Leishmania infantum chagasi expressed in Escherichia coli M15
Published in Preparative Biochemistry and Biotechnology, 2018
Luan Tales Costa de Paiva Vasconcelos, Marcos Antônio Oliveira Filho, Vitor Troccoli Ribeiro, Jaciara Silva de Araújo, Francisco Canindé de Sousa Junior, Daniella Regina Arantes Martins, Everaldo Silvino dos Santos
In a certain way, apparently, there must be a balance in which the OD600 changes that maximizes the expression of the antigen of interest. On one hand, a very low OD600 does not have a satisfactory cell amount for the level of expression desired once it is generally considered that OD600 is a partial surrogate for plasmid copy number. In this study, E. coli M15 harboring pQE-30 expression vector encoding the gene for the 503 antigen was used.[6] Thus, a higher cell density is sought, considering that the formation of the product of interest is associated with the formation of biomass, aims to maximize Pp/x. However, due to catabolic repression, the cell can “shut down” the regulatory mechanism of initiation of the lac operon as long as there is an easily assimilated carbon source available for it. Due to this cell preference, the cell density along with the depletion level of the carbon source may be the key point for the cell to allow or not the action of the promoter in order to initiate the transcription of the heterologous gene.