Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli

Sharma, Kusum; Babu, P.V. Cherish; Sasidhar, P. V. K.; Srinivas, V. K.; Mohan, V.; Krishna, Ella

doi:10.1016/j.pep.2008.02.020

Cited by 23 publications

(16 citation statements)

References 29 publications

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“…[21] Initially, a codon-optimized EGF gene, including appropriate cloning sites at both termini for insertion into the pCDFCP8 backbone was prepared by PCR-based total gene synthesis. [22] Standard molecular cloning techniques were used to introduce this fragment, preceded by a short hydrophilic spacer peptide, at the C-terminus of the CP sequence to yield pCCP8EGF.…”

Section: Resultsmentioning

confidence: 99%

Cell Targeting with Hybrid Qβ Virus‐Like Particles Displaying Epidermal Growth Factor

2011

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Structurally uniform protein nanoparticles derived from the self-assembly of viral capsid proteins are attractive platforms for the multivalent display of cell-targeting motifs for use in nanomedicine. Virus-based nanoparticles are of particular interest because the scaffold can be manipulated both genetically and chemically to simultaneously display targeting groups and carry a functional payload. Here, we displayed the human epidermal growth factor (EGF) on the exterior surface of bacteriophage Qβ as a C-terminal genetic fusion to the Qβ capsid protein. The co-assembly of wild-type Qβ and EGF-modified subunits resulted in structurally homogeneous nanoparticles displaying between 5 and 12 copies of EGF on their exterior surface. The particles were found to be amenable to bioconjugation via standard methods as well as the high-fidelity copper catalyzed azide-alkyne cycloaddition reaction (CuAAC). Such chemical derivatization did not impair the ability of the particles to specifically interact with the EGF receptor. Additionally, the particle-displayed EGF remained biologically active promoting auto-phosphorylation of the EGF receptor and apoptosis of A431 cells. These results suggest that hybrid Qβ-EGF nanoparticles could be useful vehicles for targeted delivery of imaging and/or therapeutic agents.

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Section: Resultsmentioning

confidence: 99%

Cell Targeting with Hybrid Qβ Virus‐Like Particles Displaying Epidermal Growth Factor

2011

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“…However, these studies are limited at several levels in understanding the cytoprotection and repair mechanism(s) underlying the healing process [18]. The rhEGF which has been developed indigenously by BBIL India [12] have proven to be effective in the healing of diabetic foot ulcers, skin burns and wound healing when tested in the human subjects [13]. Literature survey shows that there have been no studies undertaken to implicate the beneficial effects of rhEGF in the management of gastric antral ulcers induced with NSAID keeping in view of the fact that NSAIDs have been frequently used as anti-inflammatory drugs for arthritic patients and that gastric ulceration is widely prevalent especially in the antral region [9].…”

Section: Discussionmentioning

confidence: 99%

“…In line with these reports it has been demonstrated that orally administered EGF ameliorated the gastric and duodenal mucosa injury induced with the ethanol treatment [11]. The Recombinant Human EGF (rhEGF) developed indigenously by Bharat Biotech International Limited has been from expression of a synthetic gene in Escherichia coli similar to the natural EGF with its purity to be 99% assessed by HPLC [12]. Further, its healing functions have been unequivocally demonstrated in diabetic foot ulcers, burns and skin grafts in the human subjects [13].…”

Section: Introductionmentioning

confidence: 97%

Recombinant Human Epidermal Growth Factor Alleviates Gastric Antral Ulcer Induced by Naproxen: A Non-steroidal Anti Inflammatory Drug

et al. 2010

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BackgroundTo study the effect (s) of recombinant human Epidermal Growth Factor (rhEGF) on naproxen induced gastric ulcer in Wistar NIN rats.MethodsMale Wistar NIN rats were randomly divided into six experimental groups: Group I - Control, Group II - Naproxen treated, Group III - Naproxen with rhEGF/7 days, Group IV - Naproxen without rhEGF/7 days, Group V - Naproxen with rhEGF/14 days, and Group VI - Naproxen without rhEGF/14 days. Gastric ulcer was induced with naproxen at a concentration of 80 mg/kg by oral administration. After 24 hours of induction of ulcer, rhEGF treatment was started at a concentration of 100 µg/kg. Ulcer presence and healing were confirmed by histopathology study and molecular markers.ResultsNaproxen per se induced gastric antral ulcers in Wistar NIN rats. Compared with control rats, naproxen induced rats had increased lipid peroxide content in serum. Subsequent decrease in lipid peroxide was observed in rhEGF treated groups. Treatment with rhEGF significantly resulted in healing of the ulcers, which was evident by 7 days of rhEGF treatment with total healing seen by 14 days. Significant increase in immunoreactivity for Cox-2 was observed when compared to control groups, whereas less immunoreactivity of Cox-2 was observed in rhEGF treated group. Compared with control group, naproxen induced group exhibited more gene expression of both Cox-2 and TGF beta while gene expression of Cox-2 and TGF beta in rhEGF group was comparable to control group.ConclusionsThe beneficial effects of rhEGF in the management of ulcer healing can be understood. Oral rhEGF can promote healing of the rats with gastric ulcer by stimulating Cox-2 and TGF-beta expression.

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“…Native EGF, like many disulfide-containing proteins, accumulates in inclusion bodies when expressed into the reducing cytosolic environment of E. coli , 24,26 and addition of the cla MP Tag did not affect this outcome (unpublished data). By expressing EGF in an engineered strain that contains a more oxidizing cytoplasmic environment, proper folding is achieved (Figure 2).…”

Section: Resultsmentioning

confidence: 96%

claMP Tag: A Versatile Inline Metal-Binding Platform Based on the Metal Abstraction Peptide

Mills

Krause

et al. 2014

Bioconjugate Chem.

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Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals. Because chemical conjugation requires additional processing and purification steps and yields a heterogeneous mixture of products, inline incorporation of a peptide tag capable of metal binding is a highly preferable alternative. Development of a transition metal binding tag would provide opportunity to greatly expand metal-based analyses. The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag. The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

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Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli

Cited by 23 publications

References 29 publications

Cell Targeting with Hybrid Qβ Virus‐Like Particles Displaying Epidermal Growth Factor

Cell Targeting with Hybrid Qβ Virus‐Like Particles Displaying Epidermal Growth Factor

Recombinant Human Epidermal Growth Factor Alleviates Gastric Antral Ulcer Induced by Naproxen: A Non-steroidal Anti Inflammatory Drug

claMP Tag: A Versatile Inline Metal-Binding Platform Based on the Metal Abstraction Peptide

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