Cystathionase: high-performance liquid chromatography. Molecular cloning in lambda gt11. Nonradioactive immunodetection of fusion protein.
A method of purification of rat liver cystathionase by high-performance liquid chromatography (HPLC) utilizing non-ideal gel filtration method is proposed. Resolution factors-flow rate, pH values, ionic strength of the mobile phase-were optimized. Antibodies to the enzyme were purified using an immunosorbent synthesized on the basis of epoxylated Toyopearl-65. Radioimmunoassay and immunoblotting demonstrated antibody monospecificity towards cystathionase. These monospecific antibodies were utilized for detecting enzyme amounts (up to 30 pg) using the avidin-biotin system. Rat cDNA expression library in phage lambda gt11 was screened. The cystathionase cDNA clone was isolated, and the structure of the insert was determined.
Shuster
A M
AM
Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow.
Kvashuk
O A
OA
Chumakov
I
I
Prassolov
V S
VS
Gabibov
A G
AG
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D010582:Bacteriophage lambda; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003542:Cystathionine gamma-Lyase / Q000235:genetics / Q000302:isolation & purification*; D007163:Immunosorbent Techniques; D008190:Lyases / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis
D000906:Antibodies; D011993:Recombinant Fusion Proteins; D008190:Lyases; D003542:Cystathionine gamma-Lyase