A Novel Lysozyme Obtained By Exon Repetition In Vitro

Evolution of eukaryotes is mediated by sexual recombination ofparental genomes. The natural process of creating new combinationsof exons by introns recombination is called exon shuffling. It is theimportant method of directed evolution.Shortly after the discovery of split genes, it was realized that theexistence of introns may have important consequences on proteinevolution. It was pointed out that recombination within introns couldassort exons independently, and middle repetitious sequences inintrons may create hotspots for recombination to shuffle the exonicsequences. The presence of introns in most eukaryotic proteincodinggenes and their absence from prokaryotes was explain two types ofhypotheses: the "introns early"hypotheses and the "introns late"hypotheses.Lysozyme(EC3.2.1.17) was discovered more than eighty yearsago in 1922 by Alexander Fleming in human nose mucus andhydrolyzes the β-(1-4) glycosidic bond of the polysaccharide ofbacterial cells or chitin. Lysozyme has been extensively studied duringthe last decades. It is an historically important enzyme, being amongthe first to be sequenced, the first for which a three-demensional modelwas suggested using X-ray crystallography, and the first for which adetailed mechanism of action was proposed.The exon pattern is to some degree related to the structuralsubdivision of the final protein product . however, the relationship ofexons to functional units of lysozyme is better established. Exon 1 and4, code for the amino and carboxy-terminal regions of the enzyme,These regions increase the stability of the molecule but are not directlyinvolved in the catalytic function. Exon 3 codes for amino acids82-108, which give additional substrate specificity, determine thecleavage frame for the alternating N-acetylglucosamine/N-acetylmuramic acid chain, and increase the catalytic efficiency ofthe active center;Exon 2 codes for amino acids 28-82, which includethe catalytically active residues and a cluster of amino acids whichbind rings C, D, E, and F of the oligosaccharide substrate. It is one of the most industrially valuable enzymes, which havemany potential uses as a non-allergic anti-inflammation drug, anadditive to food, and so on. But in spite of its usefulness, at present itis mainly supplied from human milk, placenta, or the urine ofleukemia patients and the amount of supply only satisfies the demandfor laboratory use. So its production in large quantities is industriallyof great value. Sharp et al. reported the synthesis of lysozyme by thesolid phase peptide synthesis method, but the specific activity of theproduct remained low and the synthetic procedure were rathercomplicated and laborious. So we considered that the production inmicroorganisms using recombinant DNA techniques would be moreadvantageous for industrial purposes. The chemical synthesis of genesis one of the powerful techniques on the production of proteins usingrecombinant DNA techniques. The production of a protein using achemically synthesized gene becomes especially important when weintend to produce proteins whose amino acid sequences are already...