Hierarchical strategy for protein folding and design: synthesis and expression of T4 lysozyme gene and two putative folding mutants
- 1 January 1987
- journal article
- research article
- Published by Oxford University Press (OUP) in Protein Engineering, Design and Selection
- Vol. 1 (6) , 481-485
- https://doi.org/10.1093/protein/1.6.481
Abstract
A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesized and cloned by ligation of 26 deoxyribooligo-nucleotide fragments in two steps with a linearized plasmid followed by transformation. On selection by colony hybridization and DNA sequence analysis, clone pTLY.10 was identified to contain a complete T4 lysozyme synthetic DNA. On expression under lac-promoter, unfused T4 lysozyme was obtained in ˜4–6% yield. The design and synthesis of two putative folding mutants, flexible (Gly-Gly-Gly) and rigid (Asn-Asp-Gly) at position 73-74-75, were based on hierarchical principles. Both mutants lost enzymatic activity of the wildtype. These results are readily understandable if the hierarchical organization of the structure is taken into account. A possible explanation is that the catalytic sites are blocked in both mutants.This publication has 5 references indexed in Scilit:
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