Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

Abstract

Troponin I (TnI) is a muscle-specific protein and plays an allosteric function in the Ca 2+ regulation of cardiac and skeletal muscle contraction. Expression of cloned cDNA in E. 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. KeywordsTroponin I; Protein conformation; Bi-cistronic expression; Codon usage Muscle contraction is regulated by intracellular Ca 2+ via the thin filament-based troponintropomyosin system. Troponin is a striated muscle-specific protein complex contains three subunits: the Ca 2+ -binding subunit troponin C (TnC), the tropomyosin binding subunit troponin T (TnT) and the inhibitory subunit troponin I (TnI) [1,2]. During muscle contraction, cytoplasmic Ca 2+ rises and binds to TnC to induce a series of conformational changes in the troponin complex that release the inhibition of actomyosin ATPase and activate force development [3]. Three homologous TnI genes (cardiac, fast skeletal muscle, and slow skeletal muscle) have evolved in vertebrates to encode muscle-type-specific TnI isoforms [4]. Primary structures of cardiac, fast and slow skeletal muscle TnI isoforms are highly conserved. The main structural difference is a cardiac TnI-specific ~30 amino acids NH 2 -terminal extension [5].* To whom correspondence should be addressed. Tel: (847) 570-1960. Fax: (847)570-1865. E-mail: jpjin@northwestern.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered whi...