Production of a biologically active human basic fibroblast growth factor using silkworm-baculovirus expression vector system

Masuda, Akihiro; Xu, Jian; Minamihata, Kosuke; Kagawa, Genki; Hamada, Yusei; Morifuji, Yoshiki; Yano, Takumi; Hino, Masato; Morokuma, Daisuke; Karasaki, Noriko; Mon, Hiroaki; Kamiya, Noriho; Kusakabe, Takahiro; Lee, Jae Man

doi:10.1016/j.aspen.2018.05.002

Cited by 3 publications

(1 citation statement)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In recent decades, successful expression of hbFGF 146 or hbFGF 155 has been achieved in various hosts, including E. coli ( Feng et al, 2004 ; Chen et al, 2012 ; Rassouli et al, 2013 ; Soleyman et al, 2016 ), Bacillus subtilis ( Kwong et al, 2013 ; Hu et al, 2018 ), yeast ( Le et al, 2020 ), silkworm ( Masuda et al, 2018 ), and plants ( Ding et al, 2006 ; Yang et al, 2018 ). Of these, E. coli is an exemplary choice for the industrial-scale production of hbFGF due to its time-saving and cost-effective nature.…”

Section: Introductionmentioning

confidence: 99%

Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

Li,

Yu,

Lai

et al. 2024

Front. Pharmacol.

View full text Add to dashboard Cite

Introduction: Human basic fibroblast growth factor (hbFGF) is a highly valuable multifunctional protein that plays a crucial role in various biological processes. In this study, we aim to accomplish the scaling-up production of mature hbFGF (146aa) by implementing a high cell-density fermentation and purification process on a 500-L scale, thereby satisfying the escalating demands for both experimental research and clinical applications.Methods: The hbFGF DNA fragment was cloned into a mpET-3c vector containing a kanamycin resistance gene and then inserted into Escherichia coli BL21 (DE3) plysS strain. To optimize the yield of hbFGF protein, various fermentation parameters were systematically optimized using BOX-Behnken design and further validated in large-scale fermentation (500-L). Additionally, a three-step purification protocol involving CM-Sepharose, heparin affinity, and SP-Sepharose column chromatography was developed to separate and purify the hbFGF protein. Isoelectric focusing electrophoresis, MALDI-TOF/MS analysis, amino acid sequencing, CD spectroscopy, and Western blotting were performed to authenticate its identity. The biological efficacy of purified hbFGF was evaluated using an MTT assay as well as in a diabetic deep second-degree scald model.Results: The engineered strain was successfully constructed, exhibiting high expression of hbFGF and excellent stability. Under the optimized fermentation conditions, an impressive bacterial yield of 46.8 ± 0.3 g/L culture with an expression level of hbFGF reaching 28.2% ± 0.2% was achieved in 500-L scale fermentation. Subsequently, during pilot-scale purification, the final yield of purified hbFGF protein was 114.6 ± 5.9 mg/L culture with RP-HPLC, SEC-HPLC, and SDS-PAGE purity exceeding 98%. The properties of purified hbFGF including its molecular weight, isoelectric point (pI), amino sequence, and secondary structure were found to be consistent with theoretical values. Furthermore, the purified hbFGF exhibited potent mitogenic activity with a specific value of 1.05 ± 0.94 × 106 AU/mg and significantly enhanced wound healing in a deep second-degree scald wound diabetic rat model.Conclusion: This study successfully established a stable and efficient large-scale production process of hbFGF, providing a solid foundation for future industrial production.

show abstract

Section: Introductionmentioning

confidence: 99%

Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

Li,

Yu,

Lai

et al. 2024

Front. Pharmacol.

View full text Add to dashboard Cite

show abstract

Production of an active Mus musculus IL-3 using updated silkworm-based baculovirus expression vector system

Nagai

Ebihara

Kakino

et al. 2021

Journal of Asia-Pacific Entomology

View full text Add to dashboard Cite

Possibilities of Bombyx mori (B. mori) biotechnological platform for regenerative medicine

Yumatov,

Evlagina,

Evlagin

et al. 2024

РОиТ

View full text Add to dashboard Cite

Regenerative medicine aims at changing modern medicine practice by eliminating core reasons of diseases and disorders. Regenerative medicine includes gene therapy, cell therapy and products of tissue engineering that are destined for augmentation, regeneration or replacement of organs, tissues, genes and metabolic processes in the organism. Biomaterials are amongst key components of regenerative medicine on which successful strategies are based.The review of biotechnological methods implemented in the USP and DSP stages with the use of B. mori was made. The methods analysed are aimed at improving quality characteristics and obtaining new kinds of biomaterials to meet the needs of regenerative medicine and biomedicine. The diversity of biotechnological solutions that allow to gain a wide spectrum of biomaterials (incl. derivatives of cocoon shell such as fibroin, sericin and their composites; recombinant derivatives; antimicrobial peptides; modified transgenic silk fibres;transgenic fibres that contain growth factors and peptides; etc.) is a unique basis for the bioindustrial platform on the B. mori basis.

show abstract

Production of a biologically active human basic fibroblast growth factor using silkworm-baculovirus expression vector system

Cited by 3 publications

References 22 publications

Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

Production of an active Mus musculus IL-3 using updated silkworm-based baculovirus expression vector system

Possibilities of Bombyx mori (B. mori) biotechnological platform for regenerative medicine

Contact Info

Product

Resources

About