HIGH-LEVEL BIOSYNTHETIC SUBSTITUTION OF METHIONINE IN PROTEINS BY ITSANALOGS 2-AMINOHEXANOIC ACID, SELENOMETHIONINE, TELLUROMETHIONINE ANDETHIONINE IN ESCHERICHIA-COLI

We have utilized a T7 polymerase/promoter system for the high-level in corporation of methionine analogs with suitable labels for structural research (X-ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosy nthetic high-level substitution of methionine by 2-aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replac ement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X-ray structural analysis. Conditions for expression wer e optimized concerning the frequency of appearance of revertants, high -level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxo trophic for methionine, was grown under methionine-limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high-le vel incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and re suspended in fresh media without methionine; telluromethionine was add ed and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heav y atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy-atom derivatives suitable for X-ray struct ural analysis, supplementing the traditional trial-and-error preparati on of heavy-atom derivatives for the method of multiple isomorphous re placement. Furthermore, the successful high-level incorporation of ami no acid analogs can provide single-atom mutations for the detailed stu dy of the structure and function of proteins.