کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1927140 1536504 2007 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression
چکیده انگلیسی

Troponin I (TnI) is a muscle-specific protein and plays an allosteric function in the Ca2+ regulation of cardiac and skeletal muscle contraction. Expression of cloned cDNA in Escherichia coli is an essential approach to preparing human TnI and mutants for structural and functional studies. The expression level of cardiac TnI in E. coli is very low. To reduce the potential toxicity of cardiac TnI to the host cell, we constructed a bi-cistronic expression vector to co-express cardiac TnI and cardiac/slow troponin C (TnC), a natural binding partner of TnI and a protein that readily expresses in E. coli at high levels. The co-expression moderately increased the expression of cardiac TnI although a high amount of TnC protein was produced from the bi-cistronic mRNA. The use of an E. coli strain containing additional tRNAs for certain low bacterial usage eukaryotic codons improved the expression of cardiac TnI. Modifications of two 5′-regional codons that have predicted low usages in bacterial cells did not reproduce the improvement, indicating that not the 5′ but the overall codon usage restricts the translational efficiency of cardiac TnI mRNA in E. coli. However, deletion of the cardiac TnI-specific N-terminal 28 amino acids significantly improved the protein expression independent of the host cell tRNA modifications. The results suggest that the regulatory N-terminal domain of cardiac TnI is a dominant factor for the incompatibility in bacterial cells, supporting its role in modulating the overall molecular conformation.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Archives of Biochemistry and Biophysics - Volume 461, Issue 1, 1 May 2007, Pages 138–145
نویسندگان
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