Critical Factors Affecting the Success of Cloning, Expression, and Mass Production of Enzymes by Recombinant<i>E. coli</i>

Fakruddin, Md.; Mazumdar, Reaz Mohammad; Mannan, Khanjada Shahnewaj Bin; Chowdhury, Abhijit; Hossain, Md. Nur

doi:10.5402/2013/590587

Cited by 83 publications

(66 citation statements)

References 47 publications

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“…Using endogenous human exosomes as a proof of concept, we have demonstrated that the purification procedures discovered are scalable-and this can be done cost-effectively by suspension conditioning normally adherent HEK-293 cells-providing excellent starting materials for further biochemical, enzymological, and structural study. Because this approach has significant advantages over in vitro assembly of components heterologously expressed in bacteria, which suffers from innate difficulties in expressing large proteins, the need to coexpress multiple interaction partners, and the lack of endogenous post-translational modifications (Fakruddin et al 2013), we believe it will prove to be of great general utility in the study of macromolecular assemblies.…”

Section: Discussionmentioning

confidence: 99%

Purification and analysis of endogenous human RNA exosome complexes

Domanski

Upla

Rice

et al. 2016

RNA

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As a result of its importance in key RNA metabolic processes, the ribonucleolytic RNA exosome complex has been the focus of intense study for almost two decades. Research on exosome subunit assembly, cofactor and substrate interaction, enzymatic catalysis and structure have largely been conducted using complexes produced in the yeast Saccharomyces cerevisiae or in bacteria. Here, we examine different populations of endogenous exosomes from human embryonic kidney (HEK) 293 cells and test their enzymatic activity and structural integrity. We describe methods to prepare EXOSC10-containing, enzymatically active endogenous human exosomes at suitable yield and purity for in vitro biochemistry and negative stain transmission electron microscopy. This opens the door for assays designed to test the in vitro effects of putative cofactors on human exosome activity and will enable structural studies of preparations from endogenous sources.

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Section: Discussionmentioning

confidence: 99%

Purification and analysis of endogenous human RNA exosome complexes

Domanski

Upla

Rice

et al. 2016

RNA

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“…Chinese hamster ovary (CHO) cells, used for the manufacture of a wide range of heterologous proteins, require a demanding selection process to generate production clones efficiently . This is because the expression level of the heterologous gene is determined by multiple factors, including the strength and efficiency of regulatory sequences directing its transcription and processing into messenger RNA (mRNA), the chromosomal integration site, the copy number, the efficiency of translation as well as specific requirements of the particular protein such as post‐translational assembly and modification steps, and finally, secretion . In this context, promoters and their genetic control elements such as enhancers play an important role in integrating and processing gene expression …”

Section: Introductionmentioning

confidence: 99%

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis

Nguyen

Baumann

Dhiman

et al. 2019

Biotechnology Journal

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For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine‐tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5′‐ and 3′‐end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications.

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“…Recently, several recombinant therapeutic proteins and industrial enzymes are produced using E. coli expression system ( Table 1 ) (Fakruddin et al, 2013; Spadiut et al, 2014; Mane and Tale, 2015). E. coli , produces recombinant proteins, mainly three different forms such as inclusion body, the secretary as well as soluble forms (Fahnert et al, 2004; Zerbs et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review

Gupta

Shukla

2017

Front. Pharmacol.

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The protein productions strategies are crucial towards the development of application based research and elucidating the novel purification strategies for industrial production. Currently, there are few innovative avenues are studies for cloning, upstream, and purification through efficient bioprocess development. Such strategies are beneficial for industries as well as proven to be vital for effectual therapeutic protein development. Though, these techniques are well documented, but, there is scope of addition to current knowledge with novel and new approaches and it will pave new avenues in production of recombinant microbial and non-microbial proteins including secondary metabolites. In this review, we have focussed on the recent development in clone selection, various modern fermentation and purification technologies and future directions in these emerging areas. Moreover, we have also highlighted notable perspectives and challenges involved in the bioengineering of such proteins, including quality by design, gene editing and pioneering ideas. The biopharmaceutical industries continue to shift towards more flexible, automated platforms and economical product development, which in turn can help in developing the cost effective processes and affordable drug development for a large community.

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Critical Factors Affecting the Success of Cloning, Expression, and Mass Production of Enzymes by RecombinantE. coli

Cited by 83 publications

References 47 publications

Purification and analysis of endogenous human RNA exosome complexes

Purification and analysis of endogenous human RNA exosome complexes

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis

Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review

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