Physicochemical Characterization of Recombinant Human Nerve Growth Factor Produced in Insect Cells with a Baculovirus Vector

Barnett, Jim; Chow, Joan M.; Nguyen, Binh; Eggers, Daryl K.; Osen, Eric; Jarnagin, Kurt; Saldou, Natalie; Straub, Kenneth; Gu, Liangcai; Erdos, Lisa; Chaing, Hi-Shi; Fausnaugh, Jodi; Townsend, R. Reid; Lile, Jack D.; Collins, Frank M.; Chan, Hardy

doi:10.1111/j.1471-4159.1991.tb08256.x

Cited by 18 publications

(13 citation statements)

References 43 publications

(46 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similar reports have been documented for NGF and BDNF (Barnett et al, 1991;Rosenfeld and Benedek, 1993). N-terminal sequence analysis of both peaks indicated that they were identical to that of methionyl NT-3.…”

Section: Denaturation Of Nt-3 In 6 M Guanidinium Chloridesupporting

confidence: 62%

Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

Hui

Katta

et al. 1996

J Protein Chem

View full text Add to dashboard Cite

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an initial denaturation of NT-3 in 6 M guanidinium chloride (pH6) for 2 hr at 37 degrees C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110. This disulfide structure is homologous to the NGF family of neurotrophic factors.

show abstract

Section: Denaturation Of Nt-3 In 6 M Guanidinium Chloridesupporting

confidence: 62%

Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

Hui

Katta

et al. 1996

J Protein Chem

View full text Add to dashboard Cite

show abstract

“…To address these concerns, hNGF has been produced in E. coli 3233, yeast34, insect cells35363738 and mammalian cells394041. Yet in these cell systems the yield of the hNGF protein is low, and some of them, such as the E. coli and the yeast systems might be unable to provide correct post-translational modifications for hNGF.…”

mentioning

confidence: 99%

Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

Zeng

Zhu

et al. 2017

Sci Rep

View full text Add to dashboard Cite

The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins.

show abstract

“…Several attempts have been made to produce rhNGF in different systems, including Saccharomyces cerevisiae (20,21) and Escherichia coli inclusion bodies (22,23), as well as insect (24,25) and mammalian (26) cells. However, most of the available studies with rhNGF have been carried out in vitro, and the available evidence in vivo indicates that the action of rhNGF in human peripheral neuropathies was not comparable with the effect of mNGF (27).…”

mentioning

confidence: 99%

Recombinant human nerve growth factor with a marked activityin vitroandin vivo

Colangelo

Finotti²,

Ceriani³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Recombinant human nerve growth factor (rhNGF) is regarded as the most promising therapy for neurodegeneration of the central and peripheral nervous systems as well as for several other pathological conditions involving the immune system. However, rhNGF is not commercially available as a drug. In this work, we provide data about the production on a laboratory scale of large amounts of a rhNGF that was shown to possess in vivo biochemical, morphological, and pharmacological effects that are comparable with the murine NGF (mNGF), with no apparent side effects, such as allodynia. Our rhNGF was produced by using conventional recombinant DNA technologies combined with a biotechnological approach for high-density culture of mammalian cells, which yielded a production of Ϸ21.5 ؎ 2.9 mg͞liter recombinant protein.The rhNGF-producing cells were thoroughly characterized, and the purified rhNGF was shown to possess a specific activity comparable with that of the 2.5S mNGF by means of biochemical, immunological, and morphological in vitro studies. This work describes the production on a laboratory scale of high levels of a rhNGF with in vitro and, more important, in vivo biological activity equivalent to the native murine protein.mammalian cells ͉ miniPerm system ͉ neurotrophic activity R ecombinant proteins are promising for the treatment of many neurodegenerative and inflammatory diseases. Compelling basic and preclinical evidence points to neurotrophic and neurokine factors as potential candidates for preventing biochemical, pharmacological, and molecular deficits of several pathologies that lack effective therapies (reviewed in ref. 1). Some of these growth factors, including nerve growth factor (NGF) (2) have already been tested in preclinical and clinical trials. Indeed, murine NGF (mNGF) has been used successfully for human corneal and pressure ulcers (3-5), as well as vasculite (6) and crush syndrome (7), whereas recombinant human NGF (rhNGF) was shown to be effective in rodent and primate models of experimental allergic encephalomyelitis (8) and Alzheimer's disease (9, 10), as well in phase-II clinical trials of peripheral neuropathies (11). These findings are particularly interesting considering that NGF, which was identified originally as a potent neurotrophic factor for sympathetic and sensory neurons (12), was found to be essential also for basal forebrain cholinergic neurons (reviewed in ref. 13). Studies also showed that many other mammalian cells are NGF-responsive, including cells of the hemopoietic immune system (14), and, for some pathologies, symptoms also correlated with alterations of NGF serum levels (15, 16).All of this evidence supports the pharmacological interest for NGF as a useful therapeutic agent to promote the regression of many pathological conditions (1). However, the clinical efficacy of NGF has been obtained with the 2.5S mNGF (3-7), which cannot be used for human therapy on large scale. Therefore, for clinical trials and therapeutic purposes, large amounts of the recombinant human protein ...

show abstract

Physicochemical Characterization of Recombinant Human Nerve Growth Factor Produced in Insect Cells with a Baculovirus Vector

Cited by 18 publications

References 43 publications

Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

Recombinant human nerve growth factor with a marked activityin vitroandin vivo

Contact Info

Product

Resources

About