KLONIERUNG UND EXPRESSION DES TRANSFORMING GROWTH FAKTOR-(BETA)2

摘要

cDNA clones coding for TGF- beta 2 which are used to construct expression vectors capable of directing the high-level expression of mature, biologically active TGF- beta 2 in transfected Chinese Hamster Ovary cells (CHO cells) are described. The TGF- beta 2 produced and secreted by CHO transfectants has a specific activity estimated to be about half that of recombinant TGF- beta 1. The clones were isolated from a tamoxifen-treated human prostatic adenocarcinoma cell line (PC-3) using oligonucleotide probes based on the partial amino acid sequence of purified TGF- beta 2 from an African green monkey cell line, BSC-40. The cDNA sequences predict that TGF- beta 2 is synthesized as polypeptide precursor forms from which the mature 112 amino acid TGF- beta 2 subunit is derived by proteolytic cleavage. The proteins coded for by the human TGF- beta 1 and TGF- beta 2 cDNAs show an overall homology of 41%. The mature and amino- terminal precursor regions show 71% and 31% homology respectively. Northern blot analysis identified TGF- beta 2 transcripts of 4.1 kb, 5.1 kb, and 6.5 kb using mRNA from several different sources. Analysis of polyadenylated RNA from tamoxifen-treated PC-3 cells showed that these cells contain higher amounts of TGF- beta 1 transcripts than TGF- beta 2, although they produce more TGF- beta 2 protein than TDF- beta 1. This suggests that there is a post-transcriptional level of regulation for the production of these proteins.