Blue Copper Proteins: Gene Synthesis and Expression

Citation

Iverson, Sheila Anne (1988) Blue Copper Proteins: Gene Synthesis and Expression. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/z0s5-h938. https://resolver.caltech.edu/CaltechTHESIS:01222013-153725842

Abstract

A new strategy has been developed to facilitate the total synthesis of genes. The approach involves the synthesis of segments of the gene which are cloned into a vector for amplification and proof-reading. The construction of the gene proceeds from both ends toward the middle. In the synthesis, a segment of the gene is cloned into the vector and amplified; the resulting vector now contains a newly cloned segment which importantly includes unique internal restriction sites. The vector can be opened at these sites to allow insertion of an additional segment of the structural gene. A combination of three procedures: opening, cloning, amplification, constitutes a "step" of the synthesis, and a sequence of such "steps" leads to the synthesis of the total gene.

Many unique restriction sites have been designed into the gene thusly synthesized, as their presence will greatly facilitate the use of cassette mutagenesis in subsequent structure/function studies of the protein encoded by the synthetic gene.

This synthetic strategy has several attractive features. In principle it can be used for the construction of genes of essentially any length and, allows proof-reading and correction of errors at intermediate stages in synthesis. This approach thereby avoids some of the problems inherent in the more traditional strategy in which many segments of synthetic DNA are annealed, ligated together and inserted into a vector, followed by a hoped for isolation from the many species inevitably present in the resulting mixture of synthetic genetic material of a clone containing DNA with the desired sequence.

This stepwise approach has been applied to the synthesis of a gene for the blue copper protein, plastocyanin, of poplar leaf. The synthetic gene has been expressed in a fusion with a region of protein A under control of the λ_R promoter.

Thesis Files