Production and characterization of highly purified recombinant thymosin beta 4 in Escherichia coli

Li, Teng; Ma, Suyong; Tang, Xiao-Chuang; Nie, Liya; Huang, He

doi:10.1016/j.pep.2013.05.006

Cited by 7 publications

(4 citation statements)

References 13 publications

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“…So, the protein is more easily secreted into the culture medium [6, 18, 22, 23]. Glycine plays two major roles in the increasement of nanobody production: (1) increase the expression of nanobody as described before and, (2) it enhances the permeability of bacterial cell wall [5, 24]. Lower concentrations of glucose and lactose give higher production of total nanobody as reported by other researchers [6, 25].…”

Section: Discussionmentioning

confidence: 99%

Enhancement of extracellular bispecific anti‐MUC1 nanobody expression in E. coli BL21 (DE3) by optimization of temperature and carbon sources through an autoinduction condition

Rezaei

Shojaosadati

Farahmand

et al. 2020

Engineering in Life Sciences

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Escherichia coli is one of the most suitable hosts for production of antibodies and antibody fragments. Antibody fragment secretion to the culture medium improves product purity in cell culture and diminishes downstream costs. In this study, E. coli strain BL21 (DE3) harboring gene encoding bispecific anti‐MUC1 nanobody was selected, and the autoinduction methodology for expression of bispecific anti‐MUC1 nanobody was investigated. Due to the replacement of IPTG by lactose as inducer, less impurity and toxicity in the final product were observed. To increase both intracellular and extracellular nanobody production, initially, the experiments were performed for the key factors including temperature and duration of protein expression. The highest amount of nanobody was produced after 21 h at 33°C. The effect of different carbon sources, glycerol, glucose, lactose, and glycine as a medium additive at optimum temperature and time were also assessed by using response surface methodology. The optimized concentrations of carbon sources were obtained as 0.75% (w/v), 0.03% (w/v), 0.1% (w/v), and 0.75% (w/v) for glycerol, glucose, lactose, and glycine, respectively. Finally, the production of nanobody in 2 L fermenter under the optimized autoinduction conditions was evaluated. The results show that the total titer of 87.66 µg/mL anti‐MUC1 nanobody, which is approximately seven times more than the total titer of nanobody produced in LB culture medium, is 12.23 µg/L .

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Section: Discussionmentioning

confidence: 99%

Enhancement of extracellular bispecific anti‐MUC1 nanobody expression in E. coli BL21 (DE3) by optimization of temperature and carbon sources through an autoinduction condition

Rezaei

Shojaosadati

Farahmand

et al. 2020

Engineering in Life Sciences

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“…The DSB system is fundamental for bacterial physiology as it safeguards the homeostasis of the cell envelope proteome (Dutton et al., 2008; Vertommen et al., 2007). Concurrently, these folding catalysts have been invaluable tools for the production of proteins that were unattainable through mainstream purification strategies (Li et al., 2013; O’Reilly et al., 2014; Puertas et al., 2011; Suzuki et al., 2012). This has been achieved through in‐depth understanding of the biochemistry of both the DSB system and of each protein of interest and extensive trial and error.…”

Section: Discussionmentioning

confidence: 99%

“…Insertion of the dsbA open reading frame to the 5' end of a gene of interest will also effectively tag the resultant protein with the DsbA SS , thereby targeting the entire fusion to the periplasm (see section 3). DsbA has been shown to be a highly solubilizing fusion partner for many recombinant proteins (Dong et al., 2007; Fan et al., 2005; Humer & Spadiut, 2019; Kwon et al., 2011; Li et al., 2013; Lian et al., 2009; Narayanan et al., 2011; Volontè et al., 2011), some of which were previously unobtainable. The first example of the latter is bovine enterokinase (EK or enteropeptidase), a serine protease that naturally processes trypsinogen into mature trypsin (Anderson et al., 1977).…”

Section: Improving Protein Solubility By Constructing Dsba Fusionsmentioning

confidence: 99%

Harnessing the potential of bacterial oxidative folding to aid protein production

Slater

Mavridou

2021

Molecular Microbiology

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Recombinant protein production is a cornerstone of research and key to the development of diagnostics and therapeutics. From the advent of recombinant insulin production in 1982, protein expression and purification techniques have continuously improved. On average, the FDA has approved 44 protein-based therapeutics per year for the past 5 years, including monoclonal antibodies for the treatment of disease, growth factors, thrombolytics, and immunomodulators (FDA, 2020). These join a growing list of enzymes, reagents, and food additives that aid industrial and academic processes alike (Arbige et al., 2019;Pham, 2018). For the expression of these proteins, non-native expression systems offer unparalleled control on the production process compared to endogenous protein extraction protocols. This is because the user is able to tailor the cultivation parameters for each protein target by adjusting variables like the media composition, pH, agitation, aeration, temperature, cell density, inducer concentration, induction time, and feeding strategy (Ahmad et al., 2018). In particular, bacterial expression systems benefit from short doubling times, inexpensive media, genetic tractability, and ease of scaling up. Nonetheless, most bacterial laboratory strains lag behind eukaryotic expression systems in their ability to efficiently introduce posttranslational modifications (PTMs) onto a polypeptide backbone, and as such they are rapidly falling out of use. Indeed, during the 2014-2018 period, the use of non-mammalian systems for protein expression saw the largest drop on record to

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“…However, there are two main obstacles of the biosynthesis of Tb4 in E. coli: the bacterial expression of small peptides is difficult in general and this protein requires Nacetylation. Although there have been reports about the recombinant expression of human Tb4 (rhTb4), the expressions are relatively low ($1 mg/L) and the sequences are not completely consistent with natural Tb4 [29][30][31]. Some rhTb4 are expressed by fusion with the label protein, and the extra amino acids are produced after cutting, some rhTb4 are expressed in concatemer forms, and all of them are lack of N-acetylation.…”

Section: Introductionmentioning

confidence: 99%

Highly effective biosynthesis of N-acetylated human thymosin β4 (Tβ4) in Escherichia coli

Yu¹,

Cao²,

Liu³

et al. 2018

Artificial Cells, Nanomedicine, and Biotechnology

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Thymosin β4 (Tβ4) is a multifunctional N-acetylated peptide with distinct activities important at various stages. Due to its potential multiple therapeutic uses in many fields, there is an increasing need of Tβ4 at lower costs than with the use of chemical synthesis. In this research, we developed a method to produce rhTβ4 with N-acetylation in E. coli. Firstly, the E. coli strain whose chromosome being integrated by the specific N-terminal acetyltransferase ssArd1 was constructed. Secondly, the rhTβ4-Intein was constructed, in which rhTβ4 was fused to the N-terminus of the smallest mini-intein Spl DnaX. The rhTβ4 could be fully acetylated when the rhTβ4-Intein was expressed in the engineering strain. After purification, the rhTβ4-Intein fusion protein was induced with dithiothreitol (DTT) to release rhTβ4 through intein-mediated N-terminal cleavage. Under the optimal conditions, the N-terminal serine residue was shown to be 100% acetylated and the yield of N-acetylated rhTβ4 can reach 200 mg per litre. The N-acetylated rhTβ4 could be stable at 2-8 °C for 24 months in PBS buffer without protein degradation and concentration change. The N-acetylated rhTβ4 also showed the activity of binding with actins from different sources and excellent therapeutic effect on the rats with moderate to severe dry eye disease.

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Production and characterization of highly purified recombinant thymosin beta 4 in Escherichia coli

Cited by 7 publications

References 13 publications

Enhancement of extracellular bispecific anti‐MUC1 nanobody expression in E. coli BL21 (DE3) by optimization of temperature and carbon sources through an autoinduction condition

Enhancement of extracellular bispecific anti‐MUC1 nanobody expression in E. coli BL21 (DE3) by optimization of temperature and carbon sources through an autoinduction condition

Harnessing the potential of bacterial oxidative folding to aid protein production

Highly effective biosynthesis of N-acetylated human thymosin β4 (Tβ4) in Escherichia coli

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