Requirements and rationale for amber translation as pyrrolysine
David G. Longstaff · OhioLink ETD Center (Ohio Library and Information Network) · 2007
4.1Anti-(His) 6 western blot showing that expression of recombinant hexahistidine tagged mtmB1-His by M. acetivorans harboring plasmid pDL05C does not require the UTRs for the generation of the 50-kDa MtmB1-His polypeptide……………………….………… 133 4.2 Anti-MtmB immunoblot showing the expression levels of mtmB1-His in M. acetivorans transformed with pDL05C when grown on either trimethylamine or methanol…………………………… 136 4.3 Coomassie stained SDS-PAGE gel of Ni affinity purified MtmB1-His from M. acetivorans strain pDL05C grown on methanol……………… 137 4.4 Immunoblots showing the relative expression levels of mtmB1-His with the in-frame amber codon mutated to either GCA (encoding alanine), or an alternative stop codon TAA……………………………………………. 140 4.5 Anti-MtmB immunoblot showing that the mtmB1-His amber-termination product can be detected from SDS-solubilized cell extracts of M. acetivorans, harboring pDL05C, grown on trimethylamine……….. 142 4.6 Proposed mRNA secondary structure of the pyrrolysine insertion element (PYLIS) as established by chemical and enzymatic probing…………… 144 4.7 Comparison of full-length and amber-terminated polypeptides from the gene products of mtmB1-His variants with or without the PYLIS element….147 4.8 SDS PAGE gel of 5 µg of the purified MetH/MtmB1-His chimeric protein…………………………………………………………………… 150 5.1 The proposed catalytic mechanism of MtmB1………………………….163 5.2 Isolation of recombinant mtmB1-His gene product variants, expressed in M. acetivorans…..……………………………………………………… 175 xvii 5.3 Circular dichroism profiles generated with each of the MtmB1-His variants………………………………………………………………….. 179 5.4 MtmB1-His variants examined all bind MtmC, forming MtmBC complexes....................