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Protein Engineering vol. 1 no. 6 pp. 481-485, 1987
© 1987 Oxford University Press

OTHER

Hierarchical strategy for protein folding and design: synthesis and expression of T4 lysozyme gene and two putative folding mutants

S.A. Narang, Fei-Long Yao, J.J. Michniewicz, G. Dubuc, J. Phipps and R.L. Somorjai1

1Division of Chemistry, National Research Council of Canada Ottawa K1A 0R6, Canada Divisions of Biological Sciences, National Research Council of Canada Ottawa K1A 0R6, Canada

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 {small tilde}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.

Keywords: gene synthesis/expression/folding mutant

Received May 20, 1987; revised September 25, 1987;
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