Expression and purification of the h1 and h2 isoforms of calponin.
Three homologous calponin isoforms, named h1, h2, and acidic calponins, have been found in birds and mammals. Based primarily on studies of chicken gizzard smooth muscle (h1) calponin, calponin has been identified as a family of actin-associated proteins that inhibit actomyosin ATPase activity. Evolutionary divergence of the calponin isoforms suggests differentiated function. While the role of h1 calponin in smooth muscle contraction is under investigation, h2 calponin has been shown regulating the function of actin cytoskeleton. Using cloned cDNA, we expressed mammalian h1 and h2 calponins in Escherichia coli. We have developed effective methods to purify biologically active h1 and h2 calponin proteins from transformed bacterial culture. The purified calponin isoform proteins were used to generate monoclonal antibodies that reveal epitopic structure difference between h1 and h2 calponins. Together with their differential expression in tissues and during development, the structural diversity of h1 and h2 calponins suggests non-redundant physiological function. Nevertheless, h1 and h2 calponins bind F-actin with similar affinity, indicating a conserved mechanism for their role in regulating actin filaments in smooth muscle and non-muscle cells.
Jin
Jian-Ping
JP
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave, Cleveland, OH 44106-4970, USA. jxj12@po.cwru.edu
Wu
Di
D
Gao
Jimin
J
Nigam
Rita
R
Kwong
Stephen
S
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D019143:Evolution, Molecular; D005786:Gene Expression Regulation; D051379:Mice; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D020033:Protein Isoforms; D000096985:Calponins