Production of recombinant human transthyretin with biological activities toward the understanding of the molecular basis of familial amyloidotic polyneuropathy (FAP).
Transthyretin (TTR) is a plasma protein interacting with thyroxine T4 and retinol binding protein (RBP). Several variants of TTR with single amino acid substitutions have been identified as the major components of the amyloid fibrils of familial amyloidotic polyneuropathy (FAP), a fetal, autosomal dominant genetic disease. The elucidation of the molecular nature of the variants distinct from that of the wild-type TTR is crucial for understanding the amyloidogenesis in FAP, but our understanding is very poor mainly because of the unavailability of pure variant TTRs. In the present study, we used an Escherichia coli OmpA secretion vector (Ghrayeb et al., 1984) and achieved an effective production of the variant TTRs related to FAP including Met-30, Ile-33, Ala-60, Tyr-77, Met-111, and Ile-122 types. The variant TTRs produced in this system were efficiently secreted to the culture media. The chemical analysis showed that the secreted TTR (Met-30 type) has the same N-terminus as the native one. IEF analyses also indicated that the secreted product is properly processed as assessed by its pI. Furthermore, the secreted TTR was shown to have biological activities, namely, the thyroxin binding activity and the ability to associate with retinol binding protein, indicating that the secreted TTR polypeptide is properly folded. The present work also demonstrated that the processing/secretion of the recombinant TTR molecules in E. coli was strongly affected by single amino acid substitutions.
Furuya
H
H
Research Laboratory for Genetic Information, Kyushu University, Fukuoka, Japan.
Saraiva
M J
MJ
Gawinowicz
M A
MA
Alves
I L
IL
Costa
P P
PP
Sasaki
H
H
Goto
I
I
Sakaki
Y
Y
D000595:Amino Acid Sequence; D000686:Amyloidosis / Q000097:blood / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D014644:Genetic Variation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010375:Pedigree; D010523:Peripheral Nervous System Diseases / Q000097:blood / Q000235:genetics*; D011228:Prealbumin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012177:Retinol-Binding Proteins / Q000378:metabolism*; D054839:Retinol-Binding Proteins, Plasma; D013974:Thyroxine / Q000378:metabolism*
D015345:Oligonucleotide Probes; D011228:Prealbumin; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D054839:Retinol-Binding Proteins, Plasma; D013974:Thyroxine