Production and functional analysis of normal and variant recombinant human transthyretin proteins.

Abstract

The most common form of hereditary systemic amyloidosis is familial amyloidotic polyneuropathy associated with single amino acid changes in the plasma protein, transthyretin. In addition, there are two variants of transthyretin (Ser6 and Thr109) not associated with familial amyloidotic polyneuropathy but with familial euthyroid hyperthyroxinemia, also an autosomal dominant disorder. In these autosomal dominant diseases, most affected individuals are heterozygous and therefore have hybrid forms of the tetrameric plasma transthyretin. In order to study the structure/function relationships of homozygous variant transthyretins, normal human transthyretin and five variant transthyretins (Gly6----Ser, Leu58----His, Thr60----Ala, Ile84----Ser, and Ala109----Thr) were produced in Escherichia coli using the expression vector, pCZ11, and site-directed mutagenesis. These recombinant transthyretin (r-TTR) proteins showed the correct size (14 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis and self-associated into tetramers as determined by size exclusion chromatography. Recombinant normal, Ser6, and Ala60 r-TTRs had an affinity for thyroxine indistinguishable from normal human TTR purified from plasma, whereas His58 and Ser84 r-TTRs had significantly reduced affinity. On the other hand, Thr109 r-TTR had a much higher affinity, probably due to its position within the thyroxine-binding pocket. Expression of mutant transthyretins in E. coli provides the opportunity to study structure/function relationships and amyloid-forming capabilities induced by single amino acid substitutions in the transthyretin molecule.

• Authors: J R Murrell, R G Schoner, J J Liepnieks, H N Rosen, A C Moses, M D Benson
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 267 Issue 23 Pg. 16595-600 (Aug 15 1992) ISSN: 0021-9258 [Print] United States
• PMID: 1644839 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Oligodeoxyribonucleotides
• Prealbumin
• Recombinant Proteins
• Thyroxine

Topics

• Amino Acid Sequence
• Base Sequence
• Cloning, Molecular
• Escherichia coli (genetics)
• Genetic Variation
• Humans
• Kinetics
• Molecular Sequence Data
• Mutagenesis, Site-Directed
• Oligodeoxyribonucleotides
• Polymerase Chain Reaction (methods)
• Prealbumin (biosynthesis, genetics, metabolism)
• Recombinant Proteins (biosynthesis, metabolism)
• Restriction Mapping
• Thyroxine (metabolism)