1. Hierarchical strategy for protein folding and design: synthesis and expression of T4 lysozyme gene and two putative folding mutants
- Author
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J.J. Michniewicz, J. Phipps, Fei-Long Yao, Ginette Dubuc, R.L. Somorjai, and Saran A. Narang
- Subjects
Sequence analysis ,Protein Conformation ,Mutant ,Molecular Sequence Data ,Bioengineering ,Biochemistry ,chemistry.chemical_compound ,Plasmid ,Escherichia coli ,Genes, Synthetic ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Gene ,biology ,Base Sequence ,Protein engineering ,Molecular biology ,chemistry ,Gene Expression Regulation ,Oligodeoxyribonucleotides ,Chaperone (protein) ,Mutation ,biology.protein ,Protein folding ,Muramidase ,T-Phages ,Lysozyme ,Biotechnology ,Plasmids - Abstract
A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesized and cloned by ligation of 26 deoxyribooligonucleotide 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 approximately 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.
- Published
- 1987