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Genetic synthesis of repetitive polypeptides: Self-assembly of ((glycine-alanine(3))glycine-aspartate)(31) and in vivo incorporation of selenomethionine into ((glycine-alanine(3))glycine-methionine)(9)
Abstract
A series of repetitive polypeptides was synthesized in vivo from artificial genes. Target proteins were characterized to determine if chain-folded lamellar crystals could be formed and to gain insight into sequence parameters that might influence this process. The feasibility of incorporating an unnatural amino acid analog into a protein material was also investigated. The repetitive polypeptides used in these analyses were derived from a library of genetic sequences. The library was constructed by mixed-site oligonucleotide synthesis, genetic cloning and sequence analysis to yield a collection of genetic monomers encoding 19 different repetitive polypeptide sequences. Enzymatic polymerization of these genetic monomers yielded repetitive coding sequences that displayed a range of genetic stabilities. Proteins produced in vivo from these genes using inducible expression systems displayed a range of stablities as well. One library sequence, encoding octapeptide 1: $\rm\{(GlyAla)\sb3GlyAsp\}\ {\bf 1}$ was chosen for analysis of its ability to form an ordered material in the solid state. To this end the genetic monomer was isolated and polymerized enzymatically to generate a multimer representing 31 repeats of 1. The protein was expressed as a fusion in E. coli, purified and the amino-terminal fusion removed by chemical cleavage. The ability of this protein to form an ordered solid was analyzed under several conditions. Analysis by Fourier transform infrared spectroscopy and wide angle x-ray diffraction indicated the presence of two ordered conformations dependent on the solution conditions used in preparation. However, the analyses did not yield a clear picture of whether the chains assembled into the predicted lamellar crystals. Another library sequence, encoding octapeptide 2: $\rm\{(GlyAla)\sb3GlyMet\}\ {\bf 2}$ was chosen for an analysis of in vivo selenomethionine incorporation. A gene encoding nine repeats of sequence 2 was expressed as a fusion protein in an E. coli methionine auxotroph. Growth of the cells and accumulation of the protein was observed in medium lacking L-methionine but containing L-selenomethionine. Competition experiments were performed using radiolabeled methionine and unlabeled selenomethionine to determine the level of replacement by the analog. Quantitation by densitometric scanning indicates that the natural amino acid was completely replaced by the selenium-containing analog.
Subject Area
Molecular biology|Biochemistry
Recommended Citation
Dougherty, Michael John, "Genetic synthesis of repetitive polypeptides: Self-assembly of ((glycine-alanine(3))glycine-aspartate)(31) and in vivo incorporation of selenomethionine into ((glycine-alanine(3))glycine-methionine)(9)" (1993). Doctoral Dissertations Available from Proquest. AAI9329595.
https://scholarworks.umass.edu/dissertations/AAI9329595