The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli

FEBS Letters

Cross, R.A.²  Kendrick-Jones, J.²  Redwood, C.S.³  Marston, S.B.³  Bryan, J.¹ 

[1]Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

[2]MRC Laboratory of Molecular Biology, Hills Rd., Cambridge CB2 2QH, UK

[3]Natwnal Heart and Lung Institute, Dovehouse St., London SW3 6LY, UK

关键词: Caldesmon;    Bacterial expression;    Domain mapping;    DTT;    dithiothreitol;    EDTA;    ethylenediaminetetraacetic acid;    EGTA;    ethyleneglycol-bis-(β-aminoethyl ethet)N;    N;    N';    N'-tetiaacetic acid;    PIPES;    piperazine-NN-bis-2-ethanesulphonic acid;    SDS-PAGE;    sodium dodecyi sulphate polyacrylamide electrophoresis;    TAME;    p-tosyl-L-arginine methyl ester;    TPCK;    N-tosyl-L-phenylalanine chloromethyl ketone;   

DOI  :  10.1016/0014-5793(90)81233-E

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca²⁺-calmodulin and binding to actin, actin-tropomyosin, Ca²⁺-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca²⁺-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.

【 授权许可】

Unknown