Hierarchical strategy for protein folding and design: synthesis and expression of T4 lysozyme gene and two putative folding mutants.
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.
Narang
S A
SA
Division of Biological Sciences, National Research Council of Canada, Ottawa.
Yao
F L
FL
Michniewicz
J J
JJ
Dubuc
G
G
Phipps
J
J
Somorjai
R L
RL
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics*; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D011487:Protein Conformation; D013604:T-Phages / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D009113:Muramidase