Purification and characterization of recombinant tropomyosins.
The cloning of a cDNA coding for the skeletal human beta-tropomyosin in the bacterial expression vector pKK233-2 is reported. Deletion mutants were also constructed. pCF-T1088 was obtained by elimination of exon 9 and pCF-T1089 was built by deleting 2/3 of the first exon. The recombinant tropomyosins were synthesized in E. coli after induction by IPTG. The mutant proteins were characterized by western blot using antibodies raised against native tropomyosin. The amount of the human protein synthesized in E. coli varies with each mutant, suggesting the involvement of the structure of the protein or of the mRNA on the synthesis or the stability of the recombinant protein. After precipitation of most of the bacterial proteins at 100 degrees C, purification was achieved by high-performance liquid chromatography (HPLC) using TSK-DEAE, hydroxyapatite and reversed-phase columns. The chromatographic behaviour of the mutants were compared. Characterization of the mutated tropomyosins was achieved by tryptic digestion and analysis of the peptide composition by reversed-phase HPLC. A computer program for predicting the retention times of the peptides generated was written. It is shown that it is possible to identify the mutations solely by comparing the chromatogram of the tryptic digest with the profile obtained by computer simulation.
Ferraz
C
C
CRBM du CNRS, INSERM U-249, Montpellier, France.
Sri Widada
J
J
Liautard
J P
JP
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002872:Chromosome Deletion; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D004983:Ethanolamines; D006801:Humans; D006882:Hydroxyapatites; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification; D014335:Tropomyosin / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D014357:Trypsin
D004983:Ethanolamines; D006882:Hydroxyapatites; D011994:Recombinant Proteins; D014335:Tropomyosin; D014357:Trypsin; C007369:2-diethylaminoethanol