Biosynthesis of the Cyclotide MCoTI-II using an Engineered Intein

Cantor, J ; Camarero, J A

Lawrence Livermore National Laboratory

关键词: Trypsin;    Amino Acids;    Recombinant Dna;    Disulfides;    59 Basic Biological Sciences;   

DOI  :  10.2172/929172
RP-ID  :  UCRL-TR-223848
RP-ID  :  W-7405-ENG-48
RP-ID  :  929172

美国|英语

来源: UNT Digital Library

【 摘 要 】

Cyclotides are an emerging family of naturally occurring circular mini-proteins ({approx}30-40 amino acids) characterized by six conserved Cys residues (forming 3 disulfide bridges) that create a topologically unique structure designated as a cyclic cysteine knot (CCK). The cysteine knot motif, which is embedded within the macrocylic backbone, is described as two disulfide bridges that form a ring that is penetrated by the third disulfide bridge. The cyclic backbone and CCK motif together confer cyclotides with a remarkable stability and resistance to proteolytic, chemical, and thermal degradation. Further, cyclotides are functionally diverse and display a wide range of functions including uterotonic activity, trypsin inhibition, cytotoxicity, neurotensin binding, anti-HIV, antimicrobial, and insecticidal activity. Together, these characteristics make cyclotides attractive candidates for both drug design and agricultural applications, both in their native forms and as molecular scaffolds for the incorporation of novel bioactivities. [1] The ability to manipulate production of cyclotides within biological systems is critical for mutagenesis studies, production of grafted products, and the mass production of cyclotides with novel activities. My adviser's hope is to achieve this capability by employing recombinant DNA expression techniques to generate large combinatorial libraries of cyclotides. The advantage in creating a biosynthetic library (containing {approx}10{sup 6}-10{sup 10} members/library vs. chemically based libraries with typical values ranging from {approx}10{sup 3}-10{sup 5} members/library) is that it can be lead to the in vivo application of biological screening and selection methodologies based on a specific clone's ability to affect certain cellular processes.