Rabbit skeletal muscle alpha alpha-tropomyosin expressed in baculovirus-infected insect cells possesses the authentic N-terminus structure and functions.
J Muscle Res Cell Motil
; 16(2): 103-10, 1995 Apr.
Article
en En
| MEDLINE
| ID: mdl-7622625
When expressed in E. coli, skeletal muscle alpha-tropomyosin has an unacetylated N-terminus. Unacetylated alpha-tropomyosin lacks important functions; this is non-polymerizable and has a low affinity to actin. In the present work, in order to obtain fully functional recombinant alpha-tropomyosin, rabbit skeletal muscle alpha-tropomyosin (alpha-tropomyosin BV) has been expressed in baculovirus-infected insect cells. alpha-TropomyosinBV was not distinguishable from the authentic tropomyosin, not only in functional properties but also in blocked N-terminus. To know the N-terminus structure of alpha-tropomyosinBV, the N-terminal segment six amino acids long, MDAIKK, has been specifically and efficiently removed from alpha-tropomyosinBV by use of an immobilized proteolytic enzyme system based on E. coli cell bodies which carry the ompT gene product, a proteolytic enzyme localized on the outer cell wall of E. coli. The structure of recombinant alpha-tropomyosinBV was shown to be identical to the authentic protein by electrospray mass spectrometry and protein sequencing analysis. Additionally, electrospray mass spectrometry indicated a single phosphorylation not only in alpha- but also beta-tropomyosin chains in the rabbit skeletal muscle. The differentiated susceptibilities of potential ompT cleavage sites are indicative of a non-coiled-coil conformation of the N-terminus of alpha-tropomyosin.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tropomiosina
/
Proteínas Recombinantes de Fusión
/
Nucleopoliedrovirus
Límite:
Animals
Idioma:
En
Revista:
J Muscle Res Cell Motil
Año:
1995
Tipo del documento:
Article
País de afiliación:
Alemania
Pais de publicación:
Países Bajos