Reaksiyon mühendisliği prensipleriyle rekombinant insan büyüme hormonu üretimi biyoproses geliştirilmesi

Abstract

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ABSTRACTMaster ThesisBioprocess development for recombinant human growth hormone productionby reaction engineering principles%LUJÂOù(17h5.Ankara UniversityGraduate School of Natural and Applied SciencesDepartment of Chemical EngineeringSupervisor : Prof. Dr. H. Tunçer ÖZDAMARMetabolic engineering in Bacillus subtilis for extracellular human growth hormone (hGH) synthesissystem design is conceptually based on construction of a hybrid gene of two DNA fragments, i.e. signalDNA sequence of a B.subtilis extracellular enzyme gene and the DNA encoding human therapeuticprotein. Complementary oligodeoxyribo-nucleotide (oligo) primers and the polymerase chain reaction(PCR) are used to generate two DNA fragments having overlapping ends. The two DNA fragmentshaving overlapping ends are combined to form the hybrid gene in a subsequent `fusion? reaction inwhich the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3'extension of the complementary strand. Extracellular protease (subC) pre- signal DNA sequence of B.licheniformis chromosomal DNA and plasmid carrying hGH cDNA were used as templates in PCRs,respectively, for the amplification of subC signal DNA sequences and hGH mature peptide sequence.Extension of the overlaps by DNA polymerase has yielded a recombinant hybrid-gene. Hybrid genefragment was cloned into pUC19, and then sub-cloned into pMK4 E.coli- Bacillus shuttle plasmid.Thus, the new expression vector was transferred into host B. subtilis strains. Effects of oxygen-transferon product and by-product distributions in extracellular rhGH production by recombinant B. subtiliscarrying -pre(subC)