Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli.

摘要: The initial step in the conjugation of ubiquitin to substrate proteins involves activation by activating enzyme, E1. Previously, we purified and characterized multiple species E1 from wheat germ. We now describe isolation characterization a cDNA clone encoding wheat. This (UBA1) was isolated expression library with anti-wheat antibodies. It contained an open reading frame coding for 1051 amino acids directed synthesis protein that comigrated germ 117 kDa. UBA1 confirmed as (i) comparison peptide map product synthesized Escherichia coli wheat, (ii) acid sequence identity peptides generated regions derived UBA1. two additional cDNAs closely related indicated encoded small gene family Nonetheless, single poly(A+) mRNA size class 4 kilobases hybridized When expressed E. coli, catalyzed formation thiol ester linkage between carrier protein. ability containing synthesize active will allow us identify domains important function using vitro mutagenesis.