Ronald George Schoner

Structure and Function of Recombinant Human Transthyretin

In order to gain more insight into the metabolism, structure and function of transthyretin (TR) and its variants, recombinant human TR (r-TR) was synthesized rn vitro using a thermoinducible expression vector, pCZll. Normal TR cDNA was ligated into the Ndel-BamHI sites of pCZll and then transformed into E. coli JM103 cells. The r-TR showed the correct size (14kD) on PAGE and Western analysis and self associated into tetramers as seen by size exclusion chromatography. To determine proper function, iodothyronine equilibrium competitive binding assays were performed by incubating 125I-T4 with purified r-TR in the presence of increasing concentration of unlabeled iodothyronines. Affinity constants of r-TR for T4 were determined by the method of Scatchard. r-TR had the same affinity for T4 and other iodothyronine analogs (Ka=179± 16x10 Ml) as normal human TR purified from serum (Ka= 1.06±.36x10 M). Variants of TR were also studied. The rank Order of affinity of the TR variants for T was Thr >normal=Ala =Ile (heterozygous)>His-58=Tyr77 =Ser84 =Ile 1224(homozygous)=Met30 (heterozygous)>>Met 30(homozygous). These studies demonstrate that in vitro expression of human TR can provide large amounts of pure protein (normal and variant) for the study of structure/function relationships and amyloid forming capabilities.