Recent three decades have seen a significant progress in the protein engineering field. Protein engineering has not only facilitated our understanding of the sequence-structure-function relationship of proteins, but also yielded numerous invaluable new proteins or protein variants for research, medical and industrial applications. The success of protein engineering largely relies on the ability to diversify protein sequence. Protein sequence diversification is usually achieved by directed or random mutagenesis technologies. This dissertation discusses two non-canonical protein sequence diversification techniques--transposon mutagenesis and unnatural amino acid (UAA) mutagenesis.

Transposon mutagenesis is based on an in vitro transposition reaction, where a transposon DNA randomly inserts into the gene encoding the protein of interest. Removal of the transposon sequence followed by subsequent manipulation generates desired mutations. By engineering transposon DNA sequence and subsequent procedures, random "codon" deletions and substitutions can be introduced into random positions of a protein sequence. This dissertation describes how this technology can be applied to determine the minimal domain required for the fluorescence of green fluorescent protein (GFP) and improve the spectral properties of GFP.

In UAA mutagenesis, orthogonal aminoacyl tRNA and aminoacyl tRNA synthetase (aaRS) pairs are employed to reassign an amber stop codon (TAG) with an UAA. This dissertation describes how this technology can facilitate both enzymatic and nonenzymatic approaches to synthesize polyubiquitin chains.

Besides, two ongoing projects are introduced in the Appendices. The first project describes how UAA mutagenesis technology can be potentially used to genetically encode a thiotyrosine in a protein sequence, which can serve as a comparison probe. The second project intends to develop a rapid in vitro methodology to expand the functions of aaRS toward new UAAs.