Recombinant Human Nerve Growth Factor In Prokaryotic Systems In Different Expression Strategies

Nerve growth factor (NGF) is a neurotrophin that promotes growth of neurons. Human nerve growth factor not only has nutritious effects on normal nerve cells, but also has important clinical value in promoting regeneration of damaged neurons, preventing neurodegenerative diseases, blocking the growth of neural tumor and promoting the differentiation of neurons. Native NGF exists as a complex with 2βγ2 structure which combined through non-covalent bonds by three subunits, designated ,βandγ. Theβsubunit of NGF complex has the complete biological function of native NGF. Theβ-NGF is a dimer of homogeneous monomer combined through non-covalent bonds, each containing 120 amino acids and three intramolecular disulfide bonds. Currently, the pharmaceutical NGF has two mainly sources of either being extracted from submandibular gland of mouse or expressed in eukaryotic cells. Both methods have problems with low yield, high cost and hard to controllable. The E.coli has been widely used in gene engineering for producing recombinant protein with the advantages of clear genetic background, short growth period and easy control. At present, the expression of rhNGF in E.coli remains two major problems, that is, low expression level and formation of inclusion bodies, which made the study of the expression of rhNGF in prokaryotic in a stagnant situation. Therefore, it has important significance both in basic research and in clinical applications that to express rh-β-NGF highly in prokaryotic system. The main purpose of this project is to study the high or soluble expression strategy of rh-β-NGF in prokaryotic system.It is well known that there are many factors influencing the expression efficiency of interest gene in prokaryotic system, including the structure of the gene, the stability of mRNA , the initiate translation efficiency, the strength of the promoter, the choice of the expression vector, the codon bias of E.coli and the difficulty of the protein folding.During the previous study we found that the expression level of the human NGF in E.coli was very low, either using pET expression system or pBV220 expression system. The peptide is highly expressed when it was fused to a N-terminal protein such as Trx and GST but existed almost entirely in inclusion body. Exception was that when the peptide was fused to N-terminal DsbA-DsbAm, which was expressed both soluable and biologically active. Based on these findings, we implored the factors influencing the expression level and soluability of the NGF in E.coli in this thesis.Acoording to the bioinformatic analysis, we found that the sequence of humanβ-ngf contains a large number of rare codons of E.coli and the 5'end of its mRNA formats a hairpin secondary structure, which are supposed to be reasons for the low expression of rhNGF in E.coli. We designed synonymous mutant subsequently, using high frequency codon instead of rare codon, eliminating the hairpin structure of 5'end of the mRNA while maintaining the stability of its full-length mRNA. The expression level of these synonymous mutant in E.coli was confirmed to be increased than that of the wide sequence.To eliminate the hairpin strcture in the 5'end ofβ-ngf, we then designed a propeptide which composed of the leading peptide and matureβ-NGF peptide ( ProNGF ). In order to separate the mature peptide with propeptide, we inserted a thrombin cleavage site between the pro sequence and the ngf sequence. Results showed that the ProNGF can be highly expressed in E.coli with a form of inclusion body. Finally, the biologically active rhNGF was obtained after the renaturation, thrombin digestion and purification processes.The correct pairing of disulfide bond has a closely relationship to the correct folding of protein. It is reported that the disulfide bond is formed in the periplasm of E.coli. Some specialized thiol-disulfide exchanging enzymes existing in E.coli periplasm play an important role in the correct formation of disulfide bond. Dsb family contains six members of DsbA, DsbB, DsbC, DsbD, DsbE and DsbG.Disulfide bond formation protein A (DsbA) is the most oxidative protein of the thioredoxin family which directly involved in disulfide bond formation. DsbA can help protein to fold correctly both in vivo and in vitro. DsbAm (DsbA mutant protein) is a site-specific mutant of DsbA which abolished its oxidoreductase activity while maintained the solubility-aiding and localization-leading property. In this project, the two genes dsba-dsbam and ngf mutant are cloned into the prokaryotic co-expression vector pETDuet-1, and the expression of NGF was analysed in E.coli. Results showed that the NGF could be expressed in soluble form. We then purified the protein through metal chelate affinity chromatography, gel filtration and ion exchange chromatography. The biological activity of NGF was confirmed by the dorsal root ganglia assay. To be concluded, we improved the expression level and solubility of rhNGF in prokaryotic cells by modifing the codon bias, mRNA secondary sturcture and chaperons. All the work of this study will benefit the further research on the expression of rhNGF in E.coli.