Expression, purification, and in vitro cysteine-10 modification of native sequence recombinant human transthyretin.
Transthyretin (TTR) is a serum protein that is also a prominent component of deposits in two different types of systemic amyloid disease, senile systemic and familial TTR amyloidoses. Studies of recombinant TTR (rTTR) have provided many insights into the relationship between protein structure and amyloidogenicity. Yet, there is no existing recombinant system that results in high yield production of a protein that is identical in primary structure to human TTR. To date, most published studies have generated rTTR using the human gene sequence, which is poorly expressed in Escherichia coli. In addition, the gene sequence has been flanked by a 3' AUG start codon to initiate translation, resulting in the expression of a protein containing an N-terminal methionine residue not present in the human protein. We present an improved technique which can be used to generate large quantities of human native sequence TTR. Our recombinant system utilizes a gene containing codons altered for efficient expression in E. coli and an N-terminal polyhistidine tag for simplified purification. Optimization of this system was accomplished by generating a modified polyhistidine tag that was efficiently removed by dipeptidyl aminopeptidase I (DAPase). This is the first report detailing an effective and useful method for producing rTTR containing an amino acid sequence identical to human TTR. Furthermore, we describe the thiol modification of the recombinant protein to achieve exact replication of the several prominent post-translationally modified forms of TTR that have been identified in human serum.
Kingsbury
Jonathan S
JS
Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA.
Klimtchuk
Elena S
ES
Théberge
Roger
R
Costello
Catherine E
CE
Connors
Lawreen H
LH
D000686:Amyloidosis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D011228:Prealbumin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D011228:Prealbumin; D011994:Recombinant Proteins; D003545:Cysteine