Single-Stranded-Dna Sp6 and T7 Bacteriophage Promoter Plasmids for Engineering Mutant Rnas and Proteins: Extensions and Deletions at the Amino Terminus Which Alter the Activity of Preproparathyroid Hormone Signal Peptide
Mead, Alan David
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https://hdl.handle.net/2142/71445
Description
Title
Single-Stranded-Dna Sp6 and T7 Bacteriophage Promoter Plasmids for Engineering Mutant Rnas and Proteins: Extensions and Deletions at the Amino Terminus Which Alter the Activity of Preproparathyroid Hormone Signal Peptide
Author(s)
Mead, Alan David
Issue Date
1986
Department of Study
Physiology and Biophysics
Discipline
Physiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Abstract
Protein engineering systems have been developed which simplify the cloning, oligonucleotide directed mutagenesis, enzymatic sequence analysis, and expression of mutant proteins, all from a single vector. Chimeric phage-plasmid expression vectors, or phagemids, containing the intergenic region of the single stranded DNA bacteriophage f1 and either a bacteriophage SP6 or T7 promoter have been constructed. Coinfection of E. coli with these chimeric plasmids and a single stranded DNA helper phage results in the replication and secretion of the pSP6(.)f1 or pTZ plasmids as single stranded DNA viral particles. A single species of RNA can be transcribed from these vectors due to the stringent specificity of bacteriophage T7 or SP6 RNA polymerase for their respective promoter DNA sequences. Thus, these vectors are suitable for the efficient biosynthesis of large amounts of single or double stranded DNA, and translationally active RNA.
Bovine preproparathyroid hormone cDNA in both the native form and altered configurations have been inserted into these phagemids. The structure of the in vitro mutated cDNAs was determined by dideoxyribonucleotide DNA sequencing of single stranded plasmid DNA. The RNA transcribed from the cDNA constructions was efficiently translated in the wheat germ or reticulocyte cell-free systems. In the presence of dog pancreatic or chicken oviduct microsomal membranes, processing of the precursor preproparathyroid hormone to the pro form of the hormone was observed. Processing of a "stretched" version of preproparathyroid hormone that is 154 amino acids long compared to the native 115 amino acid molecule is more efficient, and this may be related to its increased length.
When the single peptide of parathyroid hormone is internalized by adding 23 amino acids to the beginning, processing to the pro-protein is severely impaired. When only 11 amino acids are added to the front of the pre-piece, conversion to the pro-protein appears normal. A deletion mutation that removes six amino acids from the N-terminus is also correctly processed. The removal of 10 or 13 residues from the N-terminus completely inhibits processing of the signal peptide. These mutations indicate a plasticity of the length and sequence of the amino terminus for membrane translocation function, within certain limits. The predicted secondary structure of the +23 mutation indicates that an additional (beta)-turn structural element not found in the +11 mutant could be responsible for the impaired processing.
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