ABSTRACT
The fused polypeptides of human epidermal growth factor and one or two S-peptide of RNase A was shown to form stoichiometric (1:1) strong noncovalent and enzymatically active complexes with S-protein of RNase A. The dissociation constants for these complexes were found to be 5.0 x 10(-7) M and 1.1 x 10(-7) M. The complexes of polypeptides with S-protein were capable to hydrolyze ribopolynucleotides, and pyrimidine-2',3'-cyclophosphates specifically, like RNase S'. A possibility was shown of effective purification of the S-peptide-containing polypeptides by affinity chromatography in which S-protein is immobilized on solid supports.
Subject(s)
Peptide Fragments/chemistry , Peptides/chemistry , Recombinant Fusion Proteins/chemistry , Ribonucleases/chemistry , Amino Acid Sequence , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Epidermal Growth Factor/chemistry , Hydrolysis , Molecular Sequence Data , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolismABSTRACT
Expression E. coli plasmid were constructed in which the human interleukin-4 (hIL4) synthetic gene is controlled by tac promoter. The expression level of the gene depends on the distance between RBS and the initial codon ATG, with the maximal production in case of the nine base pair distance. The recombinant protein, accumulated in the inclusion bodies, was solubilized, renaturated, and purified to homogeneous, biologically active preparation, the yield being 2 mg/g wet cells.
Subject(s)
Escherichia coli/genetics , Gene Expression , Genes, Synthetic , Interleukin-4/genetics , Base Sequence , Humans , Interleukin-4/isolation & purification , Interleukin-4/pharmacology , Molecular Sequence Data , Plasmids , Promoter Regions, Genetic , T-Lymphocytes/drug effectsABSTRACT
A synthetic gene coding for human interleukin-3 (hIL3) was cloned in the plasmid pTE2IL3, the gene expression being controlled by the phage fd PVIII promotor and the phage T7 gene 10 translational enhancer. Under constitutive biosynthesis conditions in E. coli, the accumulation of recombinant hIL3 (in the inclusion bodies) was up to 30-40% of the total cell protein. An effective procedure of the hIL3 isolation is suggested. The hIL3 was solubilized in 5 M guanidinium chloride, renaturated and purified to homogeneity by a single chromatographic step. The protein's yield was 34 mg/g wet cells. The isolated hIL3 showed a specific biological activity.
