Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture

Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

doi:10.1159/000441453

Cited by 11 publications

(5 citation statements)

References 24 publications

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“…Currently, the commercial bFGF is manufactured in recombinant form mainly in The high expression levels of production of recombinant human bFGF in E. coli is accompanied by problems including misfolding and the formation of insoluble aggregates known as inclusion bodies, which renders the recombinant proteins to be biologically inactive. Addressing this drawback requires extensive and complicated processing (Imsoonthornruksa et al 2015). E. coli system suffers from another di culty which is contamination to endotoxin.…”

Section: Discussionmentioning

confidence: 99%

Human basic Fibroblastic Growth Factor production by codon optimization in rice cell suspension culture

bastami¹,

Hosseini²

2022

Preprint

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Human basic fibroblastic growth factor (hbFGF), possesses a wide range of highly important biological functions, including cell proliferation, protein synthesis and metabolism. This study is the first investigation to transfer a partly home-made hbFGF gene cassette into rice cells. The human bFGF coding sequence was optimized for expression in rice in order to improve its in vitro production. Following synthesis, the expression cassette was constructed by fusing the PCR- amplified Ram3D promoter and terminator sequences in two separate steps to the optimized hbFGF CDS. Then, the cassette was transferred into the pCAMBIA1304 shuttle vector. This vector also contained a signal peptide for the secretion of the recombinant protein into the culture medium. Rice (Oryza sativa cv. Kanto 51) seeds were cultured on the 2N6PG medium composing of the N6 basal medium supplemented with 2 mg/L 2,4-D for callus induction. Callus transformation was performed by employing Agrobacterium tumefaciens strain LBA 4404. The integration of bFGF gene into the chromosomes of transgenic calli was verified by genomic DNA PCR. A transformed callus line expressing the highest level of bFGF (38.1 mg/kg FW) was recognized by ELISA and selected for the establishment of cell suspension culture. The developed transgenic rice cell culture was able to express and secrete the recombinant bFGF into the culture medium on day 3 after incubation of cells in the sucrose-free medium. The SDS-PAGE and Western blot analyses detected the bFGF in the culture medium with a molecular weight of ~17 kDa under reducing conditions. The purification of bFGF was performed using HiTrap Heparin HP column. Interestingly, the activity assay indicated that the rice derived bFGF stimulated the proliferation of NIH/3T3 cells similar to the commercial bFGF.

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Section: Discussionmentioning

confidence: 99%

Human basic Fibroblastic Growth Factor production by codon optimization in rice cell suspension culture

bastami¹,

Hosseini²

2022

Preprint

View full text Add to dashboard Cite

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“…In animal tissues the content of natural bFGF is very limited and its puri cation in quantities that meet the market demand is almost impossible (Gasparian et al, 2009). As a result, during past three decades, considerable efforts have been made for the expression of recombinant bFGF in different organisms including Escherichia coli (Andrades et al, 2001;Gasparian et al, 2009;Jia et al, 2015;Ke et al, 1992;Kwong et al, 2016;Rassouli et al, 2013;Song et al, 2013;Squires et al, 1988;Ping et al 2005;Imsoonthornruksa et al 2015;Soleyman et al 2016), Saccharomyces cerevisiae (Barr et al, 1988), Bacillus subtilis (Kwong et al, 2013), Pichia pastoris (Mu et al, 2008), Bombyx mori L. (silkworm) , rice and soybean (Glycine max) (Ding et al, 2006) seeds, and tobacco (Nicotiana tabacum) chloroplasts (Wang et al, 2015). Currently, the commercial bFGF is manufactured in recombinant form mainly in The high expression levels of production of recombinant human bFGF in E. coli is accompanied by problems including misfolding and the formation of insoluble aggregates known as inclusion bodies, which renders the recombinant proteins to be biologically inactive.…”

Section: Discussionmentioning

confidence: 99%

Human basic Fibroblastic Growth Factor production by codon optimization in rice cell suspension culture

Bastami

Hosseini

2022

Preprint

View full text Add to dashboard Cite

Human basic fibroblastic growth factor (hbFGF), possesses several highly important biological functions, such as cell proliferation, protein synthesis and metabolism. This study is the first investigation to transfer a hbFGF gene cassette into rice cells. To improve the human bFGF expression in rice an optimized coding sequence was developed. Following synthesis, the expression cassette was constructed by fusing the PCR- amplified RAm3D promoter and terminator sequences to the codon-optimized hbFGF sequence. Then, the cassette was transferred into the pCAMBIA1304 shuttle vector. This vector also contained the RAmy3D signal peptide for the secretion of the recombinant protein into the culture medium. Rice (Oryza sativa cv. Kanto 51) seeds were cultured on the 2N6PG medium composing of the N6 basal medium supplemented with 2 mg/L 2,4-D for callus induction. Callus transformation was performed by employing Agrobacterium tumefaciens strain LBA 4404. The verification of bFGF gene integration into the chromosomes of putative transgenic calli was carried out by genomic DNA PCR. A transformed callus line expressing the highest level of bFGF (38.1 mg/kg FW) was recognized by ELISA and selected to establish cell suspension culture. The expression and secretion of the recombinant bFGF into the culture medium in the developed transgenic rice cell culture was evident on day 3 after incubation of cells in the sucrose-free medium. The recombinant bFGF with a molecular weight of ~ 17 kDa was detected by the Western blot and SDS-PAGE analyses in the culture medium under reducing conditions. The purification of bFGF was performed using HiTrap Heparin HP column. The activity assay indicated that the rice derived bFGF stimulated the proliferation of NIH/3T3 cells similar to the commercial bFGF.

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“…Of these, E. coli is an exemplary choice for the industrial-scale production of hbFGF due to its time-saving and cost-effective nature. Furthermore, various fusion strategies have been employed to enhance the soluble expression of hbFGF, including the utilization of glutathione S-transferase (GST) ( Sheng et al, 2003 ; Sekiguchi et al, 2018 ), thioredoxin (Trx) ( Imsoonthornruksa et al, 2015 ; Soleyman et al, 2016 ), maltose-binding protein (MBP) ( Lemaitre et al, 1995 ), and collagen-like protein (Scl2) ( Rahman et al, 2020 ). Nevertheless, challenges such as insufficient fusion tag cleavage, high costs associated with protease usage, and prolonged processing times have hindered the implementation of these strategies in large-scale production settings.…”

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.

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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.

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Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture

Cited by 11 publications

References 24 publications

Human basic Fibroblastic Growth Factor production by codon optimization in rice cell suspension culture

Human basic Fibroblastic Growth Factor production by codon optimization in rice cell suspension culture

Human basic Fibroblastic Growth Factor production by codon optimization in rice cell suspension culture

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

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