Stepwise optimization of recombinant protein production in Escherichia coli utilizing computational and experimental approaches

Packiam, Kulandai Arockia Rajesh; Ramanan, Ramakrishnan Nagasundara; Ooi, Chien Wei; Krishnaswamy, Lakshminarasimhan; Tey, Beng Ti

doi:10.1007/s00253-020-10454-w

Cited by 32 publications

(18 citation statements)

References 100 publications

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“…The pO 2 controller is triggered by any change in pO 2 combined with a pH change. According to Packiam et al [ 18 ], a total of 500 g of glucose was used during fermentation. Since dtx protein is a hydrophobic protein, it was possible to prevent aggregation by using strains like the BL21(DE3)-derived.…”

Section: Discussionmentioning

confidence: 99%

“…Several important central elements should be considered when developing recombinant expression conditions, including the expression strain, medium form, bioprocess optimization, and mathematical modelling. The cost and reproducibility issues can be addressed by well-designed industrial scale development of one recombinant protein with optimized influential parameters and yield [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

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Expression of full and fragment-B of diphtheria toxin genes inEscherichia colifor generating of recombinant diphtheria vaccines

Abulmagd

Khattab

Zedan

2022

Clin Exp Vaccine Res

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Purpose In the present study, whole diphtheria toxin (dt) and fragment B (dtb) genes from Corynebacterium diphtheriae Park William were cloned into Escherichia coli , the purified expressed proteins were evaluated for ultimately using as a candidate vaccine. Materials and Methods The dt and dtb genes were isolated from bacterial strain ATCC (American Type Culture Collection) no. 13812. Plasmid pET29a+ was extracted by DNA-spin TM plasmid purification kit where genes were inserted using BamHI and HindIII-HF. Cloned pET29a+dt and pET29a+dtb plasmids were transformed into E. coli BL21(DE3)PlysS as expression host. The identity of the sequences was validated by blasting the sequence (BLASTn) against all the reported nucleotide sequences in the NCBI (National Center for Biotechnology Information) GenBank. Production of proteins in high yield by different types and parameters of fermentation to determine optimal conditions. Lastly, the purified concentrated rdtx and rdtb were injected to BALB/c mice and antibody titers were detected. Results The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-29a(+) carrying the dt and dtb genes was confirmed by colony polymerase chain reaction assay and were positive to grow on Luria-Bertani/kanamycin medium. The open reading frame of dt and dtb sequences consisted of 1,600 bp and 1,000 bp, were found to be 100% identical to dt and dtb sequence of C. diphtheriae (accession number KX702999.1 and KX702993.1) respectively. The optimal condition for high cell density is fed-batch fermentation production to express the rdtx and rdtb at 280 and 240 Lf/mL, dissolved oxygen was about 24% and 22% and the dry cell weight of bacteria was 2.41 g/L and 2.18 g/L, respectively. Conclusion This study concluded with success in preparing genetically modified two strains for the production of a diphtheria vaccine, and to reach ideal production conditions to achieve the highest productivity.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expression of full and fragment-B of diphtheria toxin genes inEscherichia colifor generating of recombinant diphtheria vaccines

Abulmagd

Khattab

Zedan

2022

Clin Exp Vaccine Res

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“…The bacterial growth rate, similar to other natural processes, may have countless contributing parameters. Identification and optimization of these factors pose several challenges concerning expenditure and overall economics [ 16 ]. Considering this, DoE is a powerful tool in statistical bioprocess optimization that can obtain elevated results with reduced time and effort [ 17 ].…”

Section: Discussionmentioning

confidence: 99%

Media optimization for SHuffle T7 Escherichia coli expressing SUMO-Lispro proinsulin by response surface methodology

et al. 2022

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Background SHuffle is a suitable Escherichia coli (E. coli) strain for high yield cytoplasmic soluble expression of disulfide-bonded proteins such as Insulin due to its oxidative cytoplasmic condition and the ability to correct the arrangement of disulfide bonds. Lispro is an Insulin analog that is conventionally produced in E. coli as inclusion bodies (IBs) with prolonged production time and low recovery. Here in this study, we aimed to optimize cultivation media composition for high cell density fermentation of SHuffle T7 E. coli expressing soluble Lispro proinsulin fused to SUMO tag (SU-INS construct) to obtain high cell density fermentation. Results Factors including carbon and nitrogen sources, salts, metal ions, and pH were screened via Plackett–Burman design for their effectiveness on cell dry weight (CDW) as a measure of cell growth. The most significant variables of the screening experiment were Yeast extract and MgCl2 concentration, as well as pH. Succeedingly, The Central Composite Design was utilized to further evaluate and optimize the level of significant variables. The Optimized media (OM-I) enhanced biomass by 2.3 fold in the shake flask (2.5 g/L CDW) that reached 6.45 g/L (2.6 fold increase) when applied in batch culture fermentation. The efficacy of OM-I media for soluble expression was confirmed in both shake flask and fermentor. Conclusion The proposed media was suitable for high cell density fermentation of E. coli SHuffle T7 and was applicable for high yield soluble expression of Lispro proinsulin.

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“…Recombinant protein production (RPP) is a noteworthy biotechnological technique that finds rising applications in various sectors such as healthcare, detergents, food industry, and, most importantly, in research and development [1] , [2] . Escherichia coli ( E. coli ) is considered an ideal host for RPP because it offers numerous advantages, including simple nutritional requirements, faster cellular growth, and easiness in achieving high cell densities [3] , [4] . During RPP, the protein of interest can be directed towards the periplasmic space of E. coli by the use of a short amino acid sequence called signal peptides (SP).…”

Section: Introductionmentioning

confidence: 99%

“…Despite tremendous works devoted to the aspects of RPP and technological advancement, achieving the high yields of recombinant proteins remains a challenge. Moreover, optimization of RPP involves tedious, costly, and time-consuming experiments to identify the optimal fermentation conditions which are specific to each type of protein [3] .…”

Section: Introductionmentioning

confidence: 99%

PERISCOPE-Opt: Machine learning-based prediction of optimal fermentation conditions and yields of recombinant periplasmic protein expressed in Escherichia coli

Packiam

Ooi

et al. 2022

Computational and Structural Biotechnology Journal

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Stepwise optimization of recombinant protein production in Escherichia coli utilizing computational and experimental approaches

Cited by 32 publications

References 100 publications

Expression of full and fragment-B of diphtheria toxin genes inEscherichia colifor generating of recombinant diphtheria vaccines

Expression of full and fragment-B of diphtheria toxin genes inEscherichia colifor generating of recombinant diphtheria vaccines

Media optimization for SHuffle T7 Escherichia coli expressing SUMO-Lispro proinsulin by response surface methodology

PERISCOPE-Opt: Machine learning-based prediction of optimal fermentation conditions and yields of recombinant periplasmic protein expressed in Escherichia coli

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