Purification and characterization of an exon 2-deleted human beta-tropomyosin constructed by the polymerase chain reaction.
We deleted exon 2 in human skeletal beta-tropomyosin (h beta-SK tropomyosin) using an improved adaptation of polymerase chain reaction (PCR) technology. The first PCR product was used to prime the full-length cDNA, leading to an exon 2-deleted h beta-SK tropomyosin. This new protein, des-(39-80)-tropomyosin, could then be expressed in Escherichia coli and purified to homogeneity. At the nucleotide level, the junction between exons 1 and 3 has been precisely made in the PCR product. The mutated protein was purified using high-performance liquid chromatography. Des-(39-80)-tropomyosin revealed new immunological properties but was still recognized by certain antitropomyosin antibodies. Furthermore, the structural characteristics of the mutated tropomyosin fit those of the full-length tropomyosin. This new adaptation of PCR technology appears to be suitable for every kind of mutation inside a cloned DNA molecule, and one mutation primer per mutation is sufficient.
Caron
E
E
INSERM U-249, Montpellier, France.
Ferraz
C
C
Heitz
F
F
Sr Widada
J
J
Liautard
J P
JP
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014335:Tropomyosin / Q000235:genetics* / Q000302:isolation & purification*
D011994:Recombinant Proteins; D014335:Tropomyosin; D004247:DNA