Use of protein trans-splicing to produce active and segmentally 2H, 15N labeled mannuronan C5-epimerase AlgE4

被引:12
作者
Buchinger, Edith [1 ,2 ,3 ]
Aachmann, Finn L. [2 ]
Aranko, A. Sesilja [3 ]
Valla, Svein [2 ]
Skjak-Braek, Gudmund [2 ]
Iwai, Hideo [3 ]
Wimmer, Reinhard [1 ]
机构
[1] Aalborg Univ, Dept Biotechnol Chem & Environm Engn, DK-9000 Aalborg, Denmark
[2] Norwegian Univ Sci & Technol, Dept Biotechnol, NOBIPOL, N-7491 Trondheim, Norway
[3] Univ Helsinki, Res Program Struct Biol & Biophys, Inst Biotechnol, FIN-00014 Helsinki, Finland
基金
芬兰科学院;
关键词
trans-splicing; inteins; protein ligation; alginate epimerases; DNAE INTEIN; C-5-EPIMERASE ALGE4; IN-VITRO; LIGATION; C-13; EXPRESSION; ASSIGNMENT; ALGINATE; STRATEGY; BINDING;
D O I
10.1002/pro.432
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Alginate epimerases are large multidomain proteins capable of epimerising C5 on beta-D-mannuronic acid (M) turning it into alpha-L-guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers. Attempts to study these enzymes with solution-state NMR are hampered by their size-the smallest epimerase, AlgE4, consisting of one A- and one R-module, is 58 kDa, resulting in heavy signal overlap impairing the interpretation of NMR spectra. Thus we obtained segmentally H-2, N-15 labeled AlgE4 isotopomeres (A-[H-2, N-15]-R and [H-2, N-15]-A-R) by protein trans-splicing using the naturally split intein of Nostoc punctiforme. The NMR spectra of native AlgE4 and the ligated versions coincide well proving the conservation of protein structure. The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild-type AlgE4.
引用
收藏
页码:1534 / 1543
页数:10
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