The expression and antigenicity identification of recombinant rat TGF-beta1 in bacteria.
In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.
Gao
C F
CF
Department of Laboratory Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, China.
Kong
X T
XT
Gressner
A M
AM
Weiskirchen
R
R
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D005822:Genetic Vectors / Q000502:physiology*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D053773:Transforming Growth Factor beta1
D000939:Epitopes; D011994:Recombinant Proteins; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1