Intein mediated hyper-production of authentic human basic fibroblast growth factor in Escherichia coli

Abstract

Human basic fibroblast growth factor is a functionally versatile but very expensive polypeptide. In this communication, employing a novel amplification method for the target gene and genetic optimization of a previously engineered expression construct, pWK3R, together with a refined fed-batch fermentation protocol, we report an achievement of a phenomenal yield of 610 mg/L of the 146 aa authentic human basic fibroblast growth factor (bFGF) in Escherichia coli. Construct pWK3R was first modified to form plasmid pWK311ROmpAd, which was devoid of the ompA leader sequence and possessed two copies of a DNA segment encoding a fusion product comprising an intein, Saccharomyces cerevisiae vascular membrane ATPase (VMA), and bFGF. When E. coli transformant JM101 [pWK311ROmpAd] was cultivated using the refined fed-batch fermentation protocol, superb expression resulting in a total yield of 610 mg/L of bFGF was detected. Despite existing in high levels, the bFGF remained to be soluble and highly bioactive.

Proliferation and differentiation of primary bovine myoblasts using Chlorella vulgaris extract for sustainable production of cultured meat

“…However, it must be noted that in this study, livestock‐derived sera such as FBS and HS were used to induce the proliferation and differentiation of PBMs; their use must be avoided in the future mass production of cultured meat to achieve sustainability in food production. Serum factors that induce the growth of mammalian cells can be obtained from microorganisms and are already being used commercially 7,8 . If these microorganisms are cultivated algae extracts, or serum factors can be obtained from cultivated algal extracts, it would be possible to achieve a truly sustainable food supply that is not dependent on grains or livestock serum.…”

“…Serum factors that induce the growth of mammalian cells can be obtained from microorganisms and are already being used commercially. 7,8 If these microorganisms are cultivated algae extracts, or serum factors can be obtained from cultivated algal extracts, it would be possible to achieve a truly sustainable food supply that is not dependent on grains or livestock serum.…”

“…Previously, various alternatives for serum components have been developed because of issues such as lot‐to‐lot performance variations, contamination by pathogens, and high cost. For example, growth factors such as basic fibroblast growth factor (b‐FGF) have been created from genetically modified Escherichia coli and are now commercially available 7,8 . However, as basic nutrients can be produced at a lower cost and differences between lots are less likely to appear than for serum components, substitutes for basic nutrients have not been developed as well as those for serum components.…”

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

“…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.…”

Centella asiatica (L.)-Neurodifferentiated Mesenchymal Stem Cells Promote the Regeneration of Peripheral Nerve

“…The scaffold should contain certain neurotrophic factors (NFs) [6] or viable Schwann cells [7] to achieve optimal nerve regeneration. The use of NFs could enhance nerve regeneration but also has several shortcomings, such as its high cost [8], easy degradability [9] and ability to change normal cells into malignant cells with prolonged use [10].…”