One-step production of bioactive human lipopolysaccharide binding protein from LPS-eliminated E. coli

Qiao, Jie; Dong, Ce; Wang, Xinping; Liu, Yi; Ma, Lixin

doi:10.1016/j.pep.2019.01.008

Cited by 5 publications

(3 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The main advantages of ClearColi® BL21(DE3) have generally been associated with the possibility of performing simpler downstream processing in order to obtain endotoxin-free therapeutic heterologous proteins (Lucigen 2016 ) that meet the desired quality requirements (Mamat et al 2015 ; Planesse et al 2015 ; Ueda et al 2016 ; Pooe et al 2017 ; Viranaicken et al 2017 ; Watkins et al 2017b , 2017a ; Hunt et al 2019 ; Qiao et al 2019 ; Wilding et al 2019a ). Some studies have reported better purification performance when recombinant proteins were produced using ClearColi® BL21(DE3).…”

Section: Discussionmentioning

confidence: 99%

“…To date, most of the published studies with ClearColi® BL21(DE3) have been restricted to complex medium formulations and shake flask cultures to produce enzymes (Liang et al 2015 ; An et al 2019 ; Jobin et al 2019 ; Song et al 2019 ), antigenic proteins (Martínez-Donato et al 2016 ; González-Miro et al 2017 ; Viranaicken et al 2017 ; Watkins et al 2017b , 2017a ; Abdellrazeq et al 2019 , 2020 ), and therapeutic products (Mamat et al 2015 ; Planesse et al 2015 ; Ueda et al 2016 ; Pooe et al 2017 ; Tomczak et al 2017 ; Wang et al 2017 ; Hunt et al 2019 ; Wilding et al 2019a , 2019b ; López et al 2021 ; Nguyen et al 2021 ). ClearColi® BL21(DE3) has also been used to produce diagnostic biomolecules (Masarapu et al 2017 ; Yoo et al 2017 ; Park et al 2021 ) and in specific functional studies (Moon et al 2018 ; Bong et al 2019 ; Hayashi et al 2019 ; Kobayashi et al 2019 ; Qiao et al 2019 ; Carratalá et al 2021 ; Hsu et al 2021 ; Segovia-Trinidad et al 2021 ), among other uses (Ran et al 2019 ; Sankaran et al 2019 ; Sankaran and del Campo 2019 ; Gnopo et al 2020 ). On the other hand, studies have demonstrated that the production of recombinant proteins in conventional E. coli cells is affected by several process factors (Kaur et al 2018 ) that may impact yields and titers, as well as lead to the formation of inclusion bodies and poor protein quality (Rosano and Ceccarelli 2014 ).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

Cardoso

Paredes

Campani

et al. 2022

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-β- d -thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production. Key points • ClearColi can produce as much PspA4Pro as conventional E. coli BL21(DE3) cells. • 10.5 g PspA4Pro produced in ClearColi bioreactor culture using a defined medium. • Functional PspA4Pro (98% of purity) was obtained in ClearColi bioreactor culture. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-11758-9.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

Cardoso

Paredes

Campani

et al. 2022

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…Escherichia coli BL21(DE3) and DH5α were stored in the laboratory. The pET-23a(+), pET-23a(+)-sfGFP [ 31 ] used as the expression vector were maintained in our laboratory. The plasmid pET-28a(+)-PAP-Pb coding phosphotransferase for Pseudomonas brassicacearum (PAP-Pb) was synthesized by Wuhan GeneCreate Biological Engineering Co., Ltd. (Wuhan, China).…”

Section: Methodsmentioning

confidence: 99%

Whole-Cell Display of Phosphotransferase in Escherichia coli for High-Efficiency Extracellular ATP Production

Zhao¹,

Yang²,

Xie³

et al. 2022

Biomolecules

Self Cite

View full text Add to dashboard Cite

Adenosine triphosphate (ATP), as a universal energy currency, takes a central role in many biochemical reactions with potential for the synthesis of numerous high-value products. However, the high cost of ATP limits industrial ATP-dependent enzyme-catalyzed reactions. Here, we investigated the effect of cell-surface display of phosphotransferase on ATP regeneration in recombinant Escherichia coli. By N-terminal fusion of the super-folder green fluorescent protein (sfGFP), we successfully displayed the phosphotransferase of Pseudomonas brassicacearum (PAP-Pb) on the surface of E. coli cells. The catalytic activity of sfGFP-PAP-Pb intact cells was 2.12 and 1.47 times higher than that of PAP-Pb intact cells, when the substrate was AMP and ADP, respectively. The conversion of ATP from AMP or ADP were up to 97.5% and 80.1% respectively when catalyzed by the surface-displayed enzyme at 37 °C for only 20 min. The whole-cell catalyst was very stable, and the enzyme activity of the whole cell was maintained above 40% after 40 rounds of recovery. Under this condition, 49.01 mg/mL (96.66 mM) ATP was accumulated for multi-rounds reaction. This ATP regeneration system has the characteristics of low cost, long lifetime, flexible compatibility, and great robustness.

show abstract

Application of a Novel CL7/Im7 Affinity System in Purification of Complex and Pharmaceutical Proteins

Chow

Vassylyev

2022

Methods in Molecular Biology

View full text Add to dashboard Cite

One-step production of bioactive human lipopolysaccharide binding protein from LPS-eliminated E. coli

Cited by 5 publications

References 15 publications

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

Whole-Cell Display of Phosphotransferase in Escherichia coli for High-Efficiency Extracellular ATP Production

Application of a Novel CL7/Im7 Affinity System in Purification of Complex and Pharmaceutical Proteins

Contact Info

Product

Resources

About