| Keratinocyte growth factor (KGF) was firstly isolated from the growthculture fluid of human embryo lung fibroblast by Rubin in 1989. The purifiedKGF protein was monomeric polypeptide with an apparent molecular weight of26-28 kD. The sequence of KGF cDNA revealed that KGF was a member of theFGF family. Compared with acidic FGF and basic FGF, the area of homologycomprised approximately the carboxy terminal two-thirds of the FGF codingsequence. In this region, the KGF sequence is 30%-45% identical. The KGFgene encodes a 194 amino acid protein, including a classical singal peptide forsecretion and one potential N-linked glycosylation site. At present, the cloningand expression of KGF in Escherichia coli has been finished. When expressed inbacteria, a biologically active protein was obtained with an apparent size of 21kD, including 163 amino acid residues, providing indirect evidence that thefactor synthesized in mammalian cells is glycosylated and that thispost-translation modification is not required for potent activity.In addition to human embryonic lung fibroblast, stromal cell from a varietyof other sources express KGF in culture, this includes cells from human adultlung, skin, mammary gland, stomach, bladder and prostate. γδT cells fromdermis and intestine also express KGF, but no expression was detected in adiverse groupe of epithelial cell lines, endothelial cell or melanocytes. Incontrast to other FGF, KGF's target-cell specificity was restricted to epitheliacell types. Researchers demonstrated that KGF stimulated DNA synthesis inmouse and human keratinocyte, human mammary epithelial cells, rhesusmonkey bronchial epithelial cells, rat and human prostatic epithelial cells, rathepatocytes, cornea epithelial cells, and bovine ovarian granulose cells. Thisreveals that KGF functions primarily as a paracrine mediator.At present, there is little study on KGF in China. Because of suchadvantages of genetically engineered drug as good therapeutic effect, few sideeffects, suitable for industrialized full-scale operation, to product KGF bygenetic engineering has practical importance. We chose Pichia pastoristo, one ofthe most perspective commercial tools, to express interest protein. Pichiapastoris had advantages of post-translational modification of eukaryoticexpression system and convenience, simple, low cost of prokaryotic expressionsystem as well. Most exogenous gene had higher expression level in Pichiapastoris than in bacteria, Saccharomyces cerivisiae, zooblast;Pichia pastoris haseukaryotic subcellular structures which can carry on post-translationalmodification, glycosylation, fatty acidation, protein phosphorylation;extraneousproteins were expressed and secreted efficiently in Pichia pastoris, butendogenous proteins was secreted at a low level, downstream products wereeasily isolated and purified;Pichia pastoris zymotechnique was well –developedand enlarged easily. Large scale industrialization had been established, therefore,Pichia pastoris expression system was suitable for large-scale fermentationproduction of recombination protein. At present studies on Pichia pastorisexpressing KGF protein has not been reported.In this study, we chose Pichia pastoris as bioreactor to product KGF protein,and the work we had done was showed as below: the kgf gene was cloned andinserted into pPICZαexpression vector, constructing the expression plasmidpPICZαC-kgf, which was transformed into Pichia pastoris via electroporation.We screened the transformed Pichia pastoris that highly effective, constantlyexpressed KGF by PCR and activity determination.1. Cloning of kgf cDNA sequenceWe firstly obtained the cDNA sequence encoding KGF by RT-PCR, in whichthe template RNA derives from the human embryo lung fibroblast. Then thepurified PCR production was cloned to pMD18-T vector. The results ofsequencing showed that the recombinant cloning vector pMD18-T-kgf wasconstructed correctly.2. Expression of recombinant KGF in Pichia pastorisWith the recombinant plasmid of pMD18-T-kgf as template, we designedspecific primers and obtained the sequence encoding KGF by PCR. Theendonuclease sites of XhoI and XbaI were added into the two ends of interestedsequence. Cloning the sequence to pPICZαvector after being digested withthose two endonucleases. The results of sequencing demonstrated that theconstruct was correct.we transformed purified pPICZα-kgf vector into Pichia pastoris andscreened positive clones on YPD plates containing 100μg/ml Zeocin by PCR .After 48 hours, dozens of colonies of transformants formed and recombinantKGF in Pichia pastoris was constructed.We selected several clones from YPD/Zeocin plates, induced them by inBMMY media, and analyzed the expression product by activity determinationand SDS-PAGE.Results: It indicated that the expression system is effective to express secretiontype recombinant KGF protein under the inducing function of 0.5% methanol;Through being detected the time course expression of positive clone, it wasfound that production started to increase at the first day of inducing, and reachthe peak at the day 3;on day 5 the expression of recombinant protein began toreduce;it most likely that production was degraded by proteinase in culture fluid.Analyzing the purified recombinant protein by SDS-PAGE, a protein of 28 kDamolecular weight did no appear.Under the wide PH field (3.0~6.5) Pichia pastoris can grow well and such aadvantage make it easy to improve fermentation condition. High expressionstrains selected were inoculated in media with different PH including 3.0, 3.5,4.0, 4.5, 5.0, 5.5, and 6.0, and the activity of expression production was tested.At PH 5.0 the production reached the peak, and below or above that productionexhibited reduction.To sum up, the effective expression system of recombinant KGF in Pichiapastoris had been firstly constructed by gene recombination;in this expressionsystem methyl alcohol induced the production of secreting type recombinantKGF. However, we need further improvement to increase the production andeventually make the industrial production of KGF come true.
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