Optimization of Culture Parameters and Novel Strategies to Improve Protein Solubility

Arya, Ranjana; Sabir, Jamal S. M.; Bora, Roop Singh; Saini, Kulvinder Singh

doi:10.1007/978-1-4939-2205-5_3

Cited by 12 publications

(8 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast, solubilization and refolding of protein from inclusion bodies is a common strategy although it requires denaturing conditions and a subsequent renaturing step, usually resulting in poor soluble protein recoveries [ 32 , 33 ]. Different approaches have been developed to prevent the accumulation of inclusion bodies in E. coli , such as the optimization of culture conditions, co-expression with molecular chaperones [ 34 , 35 ], lower growth temperature during gene induction [ 35 , 36 ], induction expression in early-log phase culture [ 37 ], and induction with lower levels of inducer concentration, such as Isopropyl β-D-1-thiogalactopyranoside (IPTG) [ 38 ]. In addition, several fusion tags have been developed to increase the solubility of overexpressed proteins, although with variable degrees of success.…”

Section: Resultsmentioning

confidence: 99%

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction

Cardoso

Brás²,

Costa³

et al. 2022

IJMS

View full text Add to dashboard Cite

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.

show abstract

Section: Resultsmentioning

confidence: 99%

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction

Cardoso

Brás²,

Costa³

et al. 2022

IJMS

View full text Add to dashboard Cite

show abstract

“…The commonly used molecular chaperones in E. coli are GroEL, GroES, DnaK, DnaJ and Trigger factor ( Table 2). These cytoplasmic chaperones can be employed either individually or in combination of different chaperones to enhance protein solubility and prevent formation of inclusion bodies [10,26,28,29]. The GroEL-GroES chaperone combination is highly efficient to enhance protein refolding and also prevent protein degradation.…”

Section: Plasmids Carrying Molecular Chaperones For Optimization Of Pmentioning

confidence: 99%

“…Plasmids are very commonly used as vectors in the field of genetic engineering for the purpose of cloning and expression of desired genes. Various types of plasmids are now available commercially for cloning and expression of foreign genes in a wide variety of host including E. coli, yeast and mammalian cells [10]. The desired genes to be cloned and expressed are inserted into suitable plasmid.…”

Section: Introductionmentioning

confidence: 99%

“…The cells that contain the introduced plasmid will be able to survive and grow in selective medium as they carry the plasmid with the antibiotic resistance gene. This strategy is employed to clone and express heterologous proteins in E. coli for the large-scale production of recombinant proteins for therapeutics and wide variety of functional studies [10]. In this chapter, we summarize the recent technological advancements in the field of molecular biology that have extended the use of the E. coli expression system to produce more complex proteins, including glycosylated recombinant proteins and therapeutic antibodies.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Plasmids for Optimizing Expression of Recombinant Proteins in E. coli

Al-Hejin¹,

Bora²,

Ahmed³

2019

Plasmid

View full text Add to dashboard Cite

Plasmids are important vectors for the transfer of genetic material among microbes. The transfer of plasmids causes transmission of genes involved in pathogenesis and survival, to the host bacteria leading to their evolution and adaptation to diverse environmental conditions. A large number of plasmids of varying sizes have been discovered and isolated from various microorganisms. Plasmids are also valuable tools to genetically manipulate microbes for various purposes including production of recombinant proteins. Escherichia coli is the most preferred microbe for production of recombinant proteins, due to rapid growth rate, cost-effectiveness, high yield of the recombinant proteins and easy scale-up process. Several plasmids have been designed to optimize the expression of heterologous proteins in E. coli. In order to circumvent the issues of protein refolding, the codon usage in E. coli, the absence of post-translational modifications, such as glycosylation and low recovery of functionally active recombinant proteins, various plasmids have been designed and constructed. This chapter summarizes the recent technological advancements that have extended the use of the E. coli expression system to produce more complex proteins, including glycosylated recombinant proteins and therapeutic antibodies.

show abstract

“…8 Inclusion body formation is a common issue in recombinant protein production 9 and is the result of an unbalanced equilibrium between protein precipitation and protein refolding. 10 Several strategies have been suggested to avoid inclusion body formation in E. coli, 11 including low-copy-number vectors, weak promoters, 12 co-expression of chaperones, 13 low isopropyl -D-1-thiogalactopyranoside (IPTG) concentration, low-temperature growth and induction, 14 nutrient feeding during the induction phase, and medium component optimization. 15 All these strategies harness different ways to reduce the recombinant gene expression rate and facilitate correct protein folding.…”

Section: Introductionmentioning

confidence: 99%

Soluble recombinant pyruvate oxidase production in Escherichia coli can be enhanced and inclusion bodies minimized by avoiding pH stress

Zhang

2019

J of Chemical Tech & Biotech

View full text Add to dashboard Cite

BACKGROUND: Recombinant pyruvate oxidase (pod) is in high demand owing to its broad application in biochemical analysis and low yield from wild microbial strains. Recombinant Escherichia coli harboring pET28a-pod is an efficient producer of pod, facilitating its production in high yield. Optimizing cultivation using shake flasks is necessary for high-level soluble pod production. RESULTS:During recombinant E. coli cultivation, 96.9% of pod activity was lost, and 3.1% of residual pod activity remained in a 25 mL working volume in 250 mL flasks owing to accumulation of pod in insoluble inclusion bodies. pH stress was the factor responsible for this phenomenon based on investigating dissolved oxygen and carbon source addition. Residual pod activity was increased to 43.3, 88.0 and 61.5% by maintaining the pH at 5.0, 6.0 and 7.0 respectively during the induction phase, which decreased inclusion body formation and increased production of soluble pod. Glycerol addition increased the cell density and volumetric pod activity to 21 243.3 U L −1 at pH 6.0 after 64 h induction. CONCLUSION: Soluble pod is converted to inclusion bodies under pH stress conditions, and this can be avoided by pH maintenance.

show abstract

Optimization of Culture Parameters and Novel Strategies to Improve Protein Solubility

Cited by 12 publications

References 31 publications

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction

Plasmids for Optimizing Expression of Recombinant Proteins in E. coli

Soluble recombinant pyruvate oxidase production in Escherichia coli can be enhanced and inclusion bodies minimized by avoiding pH stress

Contact Info

Product

Resources

About