Cloning, Expression, Purification, Characterization And Application Of Recombinant Human Nerve Growth Factor

Nerve Growth Factor (NGF) was first discovered and purified in the 1950s by Rita Levi-Montalcini and Stanley Cohen. NGF is involved in the growth, survival and differentiation of specific nerve cell populations. Animal tests and human phase-II trials indicated that rhNGF may be an effective treatment for diabetic and HIV-related neuropathies, NGF isolated from the mouse submaxillary gland has been used to treat cranial and peripheral nerve damage in clinics in China , but many adverse reactions have been observed . The failure of rhNGF in a large-scale phase-Ill trial of diabetic clinical trials has been attributed to inadequate dosing levels and the potential need for multiple neurotrophic factors for efficacious treatment. Further study of the clinical effects of hNGF requires a relatively inexpensive source of recombinant NGF protein. NGF produced by genetic engineering in E.coli could solve this problem since the mature protein is not glycosylated to any appreciable extent.hNGF has been expressed in E.coli as an insoluble form in inclusion bodies and as a secreted form in the E.coli periplasmic space . Isolation from inclusion bodies requires solubilization followed by refolding. Thisprocess can be inefficient and difficult because the β-subunit of hNGF contains three intrachain disulfide bonds, the proper formation of which is essential for biological activity. We designed a thioredoxin hisA/ rhNGF fusion protein to obtain efficient expression and purification of rhNGF. Thioredoxin fusion proteins are usually soluble in the E.coli cytoplasm and also induce proper protein folding and disulfide bond formation in the fusion partner. The inclusion of a HisA fragment allows efficient purification of the fusion protein using metal affinity chromatography. Using this strategy, the expressed rhNGF not only avoids the problems associated with inclusion body denaturation and refolding but also maintains biological activity.Fusion proteins represent a viable path to a therapeutic product, as illustrated by the 1997 U.S. FDA approved of the bioengineered recombinant drug interleukin 11 (rhIL-11, Neumega^TM, the generation of which employed fusion protein expression. A fusion protein approach was also used in the genesis of recombinant parathyroid hormone (PTH), which is also approved for clinical use in China.First, we synthesized hNGF gene, then the fragment was inserted into the expression vector PthioHisA, The positive clone was screened and sequenced. The synthesized hNGF gene sequence was consistent with the published sequence. The vector pThioHisA contained a Ptrc promoter, a thioredoxin leader peptide, an aspA terminator and an Amp antibiotic-resistance gene sequence. The hNGF cDNA was cloned into vector pThioHisA. Recombinant plasmid pTHNGF was transformed intoE.coli TOP10 to obtain the bioengineered strain pTHNGF/ TOP10. The plasmid was extracted for identification test by restriction endonuclease analysis. The 370 bp rhNGF cDNA fragment was detected.Second? Fermentation of bio-engineered rhNGF strain on pilot scale was studied. We used 2% vol seed culture to inoculate in LB (Amp+) culture media in a 40L fermenter for a high-density incubation at 37°C until the OD600 was¹5. Isopropyl P-thiogalactoside (IPTG) was added to a concentration of 0.5mmol/L for induction. The culture after induction was kept at 37°C for 5 hrs until the OD600 reached 40. Fermentation conditions were optimized empirically by varying the following: inoculation quantity, components of culture media, duration of induction, concentration of induction reagent, speed of nutrient supply, and the amount of dissolved oxygen during high density fermentation. Protein synthesis was induced for five additional hours for a total incubation time of approximately 10 hrs.Bacteria were collected, rinsed, and assayed by SDS-PAGE. Compared with the blank control sample, the induced bacteria sample revealed a dark band at 26kD consistent with the relative molecular weight of hNGF fusion protein. Densitometric scanning showed that the 26kD protein band represented about 15% of total protein.Third, we investigated Pilot scale of rhNGF purification. Preliminary. isolation process is as below. Osmotic shock was used to release the fusionprotein. Bacteria were collected and rinsed with buffer containing 20%sucrose, 1 mmol/L EDTA, 20mmol/L Tris.Cl, at pH8.5. Osmotic shockbuffer (20 mmol/L Tris.Cl, pH8.5) was then added to the bacteria, andstirred at 4°C for 20min. The solution was centrifuged at 10000 rpm for 30min. The supernatant was collected and concentrated by ultra-filtration. The amount of fusion protein appeared to reach approximately 20% of the total amount of protein in the cytoplasm. Purification process is as below. A pilot-scale purification protocol for hNGF was developed and optimized resulting in the following processes: Ni-chelating affinity chromatography separation —? G-25 desalting —> Sepharose-DEAE separation —*â–  G-25—?Enterokinase (EK) cleavage—> desalting —? Sepharose-CM separation -? Sephacryl S-100 separation. This strategy resulted in an overall yield of 20-30 mg/L of hNGF from the 80L experiment. To optimize Enterokinase (EK) cleavage, the temperature and time, and the proportion of enzyme and protein, were varied using orthogonal combinations. The results revealed that the most suitable conditions for Enterokinase (EK) cleavage were: 1U EK/mg protein, 32°C for 14 hrs.Fourth, biochemical and biological characterization of rhNGF was studied. The characteristics of finished rhNGF were as follows: Protein content (Lowry Assay) was lmg/ml;purity (SDS-PAGE electrophoresis and HPLC) was > 95%;Molecular weight (Mass Spectrum) was 13.2KD;ultraviolet spectrogram was 280nm;isoelectric point was 8.8-9.6;N-terminal amino acid sequencing result was consistent with the published data. Identity test (Western blot) showed positive. Content of bacterial endotoxin was less than 5EU/m;and solution pH was 6.8. Biological assay: The biological activity of rhNGF in vitro was determined with chicken dorsal root ganglia grown in vitro for 1-2 days. In assays against a Chinesereference standard (1000 IU/ml, National Institute For The Control of Pharmaceutical and Biological Products, China), the specific activity of the recombinant protein was 1.2><105U/mg. Biological activity of rhNGF in vivo was assessed in animals with hindlimb weakness from acrylamide neuropathy. Test 1 shows the effects of increased landing foot spread in animals treated with acrylamide (at time of final dose) vs. animals treated with saline. Test 2 showed Mean landing foot-spread of rats treated daily with saline (control group) or acrylamide (modeling group). Animals were suspended by the tail, black ink applied to the soles of the hind feet, and the animal dropped onto absorbent paper. The distance between hindlimbs was taken as the landing foot spread. Each animal was tested 3 times and the average foot spread per animal used to compute the mean foot spread.By the way, we setted up pathological changes experimental mode that the sympathetic nerve damnify mouse milieu nerve to test the residue of noradrenalin in the mouse submaxillary gland.The result shows the external rhNGF has the stronger protection to the light trauma sympathetic nerve, comparing with the sever trauma nerve damaged by the large dose of 6-OHD. Furthermore, we concluded that the external given rhNGF could facilitate restore of local nerve traumas, the experiment that conducted by establishment of mouse anoxemic pathological changes mode reveals the NGF has the crucial protection from the anoxemic nerve traumasWe also established the mouse dementia experimental mode, after the treatment of the mouse whose fimbria of hippocampus was transected, we find that the rhNGF could facilitate the regenesis of Cholinergic neurowhich in the injured mouse brain complex body,and it could accelerate amelioration of memorization and study.The pharmacokinetics and tissues distribution experiment of rhNGF indicated the blood concentration peak arise at 28th minutes after the mouse muscular injection of rhNGF, the blood concentration would decline according to time.the chart of blood concentration to time meet with the character of two compartment distribution model Pharmacokinetics, the rhNGF distribution in mouse according to AUC is thyroid gland,plasma, submaxillary gland, Postganglionic Neurons,adrenal gland and kidney, little in the other tissues,it shows rhNGF have much function in ganglions.the short time of elimination half-time of the rhNGF in the mouse maybe relevant to the excretion period.The results showed that mean landing foot-spread of the acrylamide-dosed groups was significantly greater than that of the control group before and after rhNGF therapy. There was no significant difference between low dosed group and the modeling control group (P>0.05).Although, the marketing of NGF isolated from the mouse submaxillary gland has been approved by the Chinese State Food and Drug Administration, hNGF, produced using genetic engineering techniques has failed to show sustained efficacy in a variety of clinical trials. Human NGF has been tested in treatments for traumatic nerve damage, peripheral neuropathy, diabetic neuropathy, HIV-associated sensory neuropathy and also for nerve damage caused by chemical and drug toxins. The pharmacokinetics, pharmacology and toxicology of hNGF have also beenstudied. The failure of rhNGF to show substantial efficacy in clinical trials has generally been attributed to its short half-life or the absence of other complementary growth factors.Large quantities of a functionally active recombinant protein are required to pursue clinical trials of NGF. Although hNGF is not glycosylated to an appreciable extent, the three intrachain disulfide bonds and the unique structure of NGF, make it difficult to obtain bioactive hNGF from E.coli. Studies with rat pheochromocytoma cell cultures (PC-9) revealed that mNGF has significant biological activity. The specific biological activity of rhNGF, however, was 5-10 times lower than that of mNGF. The reduced biological activity of rhNGF when expressed in inclusion bodies in E.coli was attributed to difficulties in the denaturation, solubilization, and refolding processes. Although, expression of a prohNGF seemed to facilitate correct folding, the protein still had to be solubilized and refolded with an overall yield from these steps of only 35%.This paper describes the expression of hNGF as a thioredoxin fusion protein containing a His A sequence. The presence of thioredoxin resulted in the production of a protein that was soluble in the E.coli cytosol. Expression as a soluble protein obviated the need for solubilization and refolding. The presence of a HisA sequence facilitated purification using chelation chromatography. The engineering of an enterokinase site and the optimization of cleavage resulted in high yields of bioactive rhNGF. The positive effect of rhNGF therapy on landing foot-spread in murine acrylamide neuropathy suggests that rhNGF is useful as a therapeuticprotein. It should be noted, however, that NGF a priori should promote the regeneration of only sensory nerve fibers and have no effect on motor nerve fibers, both of which are commonly involved in peripheral neuropathies of nutritional, metabolic, toxic and other causes. Various steps in the process described in this paper have been optimized and the entire process appears capable of large-scale production of rhNGF. The overall strategy was successful in the pilot-scale production of rhNGF and will facilitate future clinical studies in the development of rhNGF.