[New methods of isolating recombinant proteins].
Recombinant proteins are isolated from very complex protein mixtures present in the producing cell. The isolation process involves in general four mutually interconnected stages: 1. release of the recombinant protein from the cellular environment, 2. preparation of the specimen for separation, 3. separation, 4. qualitative and quantitative analysis of the preparation. Each of these stages is formed by a complex series of methods which destroy the cellular wall, solubilize the specimen and involve the use of suitable precipitation, chromatographic, electrophoretic, immunochemical and other techniques for separation and analysis. In the submitted paper the authors describe a relatively simple isolation of recombinant peptide used for the preparation of a diagnostic kit for AIDS. From knowledge of the sequence of nucleotides in cDNA antisense peptides can be derived which have a high affinity with the isolated protein and they can be also used for affinity chromatography. An effective isolation technique is the use of mimetic ligands on the basis of textile dyes. By their combination and possible modification it is also possible to achieve separation of the required protein from contaminating substances. The mentioned highly specific methods can be combined with classical chromatographic techniques. During every step individual chromatographic fractions are tested by SDS electrophoresis in polyacrylamide gel incl. possible use of immunoblotting a specific staining.
Suttnar
J
J
Ustav hematologie a krevní transfúze, Praha.
Dyr
J E
JE
Fortová
H
H
Liska
V
V
Vonka
V
V
D004926:Escherichia coli / Q000378:metabolism; D015683:Gene Products, gag / Q000302:isolation & purification; D015488:HIV Antigens / Q000096:biosynthesis / Q000302:isolation & purification; D008722:Methods; D011994:Recombinant Proteins / Q000302:isolation & purification*
D015683:Gene Products, gag; D015488:HIV Antigens; D011994:Recombinant Proteins
Nové způsoby izolace rekombinatních bílkovin.