Molecular Clone And Expression Of Nerve Growth Factor (NGF) In Pichia Pastoris,Determination Of Biological Activity

Nerve growth factor (NGF) was discovered almost half a centuryago, first in snake venom and later in mouse submaxillary glands. NGF wasdiscovered when mouse sarcoma tissue transplants in chicken embryoscaused an increase in the size of spinal ganglia. In the course of attemptingto characterize the agent responsible for this action, snake venom,employed as a phosphodiesterase, was found to be a rich source of NGF.An homologous tissue, the submaxillary gland of adult male mice, hasbecome the preferred source of NGF; other unusually large concentrationsare found in the guinea pig prostate gland and in bovine seminal plasma.The physiological relevance of these sources is not established. Nervegrowth factor is the prototype for the neurotrophin family of polypeptideswhich are essential in the developments and survival of certain sympatheticand sensory neurons in both the central and peripheral nervoussystems .The biological function of Nerve Growth Factor is themaintenance and survival of the peripheral and central nervous systems,which makes it of great therapeutic interest for the treatment of a numberofneurodegenerative diseases, the promotion of neuron regeneration ,thegrowth blockage of neural tumor . The structure reveals that the NGF monomer has an elongated shapewith the central part of the molecule formed by two pairs of twisted ,antiparallel β-strands. There are three hairpin loops on one end, and theother end carries a cysteine-knot motif that stabilizes the fold and locks themolecules in their conformation .In the biologically active form , twomonomers are arranged in a parallel manner to form a close-packedhomodimer. Pichia pastoris has been developed to be an outstanding host for theproduction of foreign proteins since its alcohol oxidase promoter wasisolated and cloned; its transformation was first reported in 1985.Compared to other eukaryotic expression systems, Pichia offers manyadvantages, because it does not have the endotoxin problem associated withbacteria nor the viral contamination problem of proteins produced inanimal cell culture. Furthermore, P. pastoris can utilize methanol as acarbon source in the absence of glucose. The P. pastoris expression systemuses the methanol-induced alcohol oxidase (AOX1) promoter, whichcontrols the gene that codes for the expression of alcohol oxidase, theenzyme which catalyzes the first step in the metabolism of methanol. Thispromoter has been characterized and incorporated into a series of P.pastoris expression vectors. Since the proteins produced in P. pastoris aretypically folded correctly and secreted into the medium, the fermentation ofgenetically engineered P. pastoris provides an excellent alternative to E.coli expression systems. The purpose of my work was to construct a stable expression systemof NGF in GS115 by genetic engineering .The main work was asfollowing: 1. A SnaBⅠand NotⅠrestriction sites were introduced into NGF generespectively by PCR .After being amplified by PCR,this NGF genecontained SnaB Ⅰ and Not Ⅰ restriction sites at its 5'and 3'endsrespectively. 2. Construction of expression plasmid pPIC9K-NGF. PCR productsdigested by SnaB Ⅰ and Not Ⅰ were inserted into the correspondingrestriction site in the expression plasmid pPIC9K. 3. pPIC9K-NGF was transformed into E.coli DH5α.The positivecolony was screened and detected by PCR. 4. The sequence of coned NGF gene was confirmed with the dideoxychain-termination method . 5. Plasmid pPIC9K-NGF was electrotransformed into GS115. 6. The positive NGF colony was again isolated and screened by PCR. 7.The NGF were expressed in GS115 under the induction of methanol.The products were identified by Tricine-SDS-PAGE. 8. Chicken embryonic dorsal root ganglion(DRG) were departed andcultured, NGF was added in the culture and its effect was noted. Recombinant plasmid pPIC9K-NGF were successfully constructedand NGF was efficiently expressed as a secretive protein in GS115. Itsmolecular weight, with 1.4×104 dalton, was in correspondance withtheoretic value. Single band of NGF was watc...