Applications, and Efficient Large-Scale Production, of Recombinant Human Epidermal Growth Factor

Wong, Wan Keung; Lam, E.; Huang, RC; Wong, Roderick; Morris, Christina

doi:10.1080/02648725.2001.10648008

Cited by 20 publications

(7 citation statements)

References 54 publications

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“…Initially, we aimed to identify the most effective strategy to mediate the periplasmic localization of the recombinant human epidermal growth factor (hEGF). This small, 53 amino acid mitogen has various (potential) applications in wound healing and is difficult to produce at industrial scale in E. coli due to the presence of three disulphide bonds in the native protein [19]. To direct hEGF to the periplasm, fusion to various signal sequences for routing via different E. coli inner membrane targeting and translocation pathways was explored (Fig.…”

Section: Resultsmentioning

confidence: 99%

Application of an E. coli signal sequence as a versatile inclusion body tag

et al. 2017

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BackgroundHeterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form.ResultsWhen screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins.ConclusionsWe present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0662-4) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Application of an E. coli signal sequence as a versatile inclusion body tag

et al. 2017

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“…Both in vivo and in vitro data have demonstrated the efficacy of EGFs in enhancing wound healing (8–11). The previous trials demonstrated that EGF healed diabetic foot ulcers faster than placebo (5,6,7) and increase collagen content of the wound (7).…”

Section: Discussionmentioning

confidence: 99%

The efficacy and safety of epidermal growth factor in treatment of diabetic foot ulcers: the preliminary results

Tuyet

Quynh²,

Minh

et al. 2009

International Wound Journal

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“…Due to its role in the regulation of cell proliferation (Boonstra et al, 1995;Carpenter and Cohen, 1990), there are numerous clinical applications for EGF, including wound healing (Robson, 1997). However, large scale production remains cost prohibitive, hampering further pharmaceutical development and clinical use (Wong et al, 2001). Detailed studies of EGF folding in vitro have shown that near quantitative yields can be achieved using long reaction times (10 h) and high concentrations of glutathione (0.25 mM GSSG and 0.5 mM GSH) (Chang et al, 2001).…”

Section: Human Epidermal Growth Factormentioning

confidence: 98%