Production of recombinant human beta2-microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis.
Amyloid of beta2-microglobulin (beta2m) origin can be diagnosed using 131I-radiolabelled-beta2m scintigraphy in patients with uremia and hemodialysis treatment. As the tracer beta2m is isolated from another patient, it carries the common risks, including viral infections such as Hepatitis B, C and HIV, which are associated with human plasma products. In order to exclude these risks we have produced recombinant human beta2m (rhbeta2m) in Escherichia coli. The expression vector pASK40DeltaLbeta2m(His)5 contains a C-terminal (His)5-tag for purification via immobilized metal ion affinity chromatography (IMAC). Size exclusion chromatography on a Superose 12 column represents the second step of purification. The isolated rhbeta2mH5 reacted in an immunochemically identical manner to native human beta2m, and showed a single band of approximately 11.8 kDa in Western blot analysis and revealed a single spot in two-dimensional gel electrophoresis. Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da. Uniformity was further proven by crystallization and N-terminal amino-acid sequence analysis. The rhbeta2mH5 protein was then produced under conditions that allow the intravenous use in humans. Intraveneously applied indium-111-labelled rhbeta2mH5 was monitored in hemodialysed patients with and without known beta2m-amyloidosis. The tracer was localized specifically to particular areas known to contain amyloid. Thus, this rhbeta2mH5 preparation is suitable for detecting amyloid-containing organs of the beta2m-class in vivo and fulfils the requirements of a tracer for common use. Finally, the use of indium-111 instead of iodine-131 has reduced the radioactive load and resulted in higher resolution.
Linke
R P
RP
Max-Planck-Institut für Biochemie, Martinsried, Germany. linke@biochem.mpg.de
Schäeffer
J
J
Gielow
P
P
Lindner
P
P
Lottspeich
F
F
Plückthun
A
A
Weiss
E H
EH
D000686:Amyloidosis / Q000150:complications / Q000000981:diagnostic imaging*; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007205:Indium Radioisotopes; D007457:Iodine Radioisotopes; D008297:Male; D008875:Middle Aged; D008970:Molecular Weight; D004369:Pentetic Acid / Q000031:analogs & derivatives; D011786:Quality Control; D011877:Radionuclide Imaging; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D006435:Renal Dialysis; D014511:Uremia / Q000150:complications / Q000000981:diagnostic imaging*; D001613:beta 2-Microglobulin / Q000096:biosynthesis* / Q000235:genetics
D017931:DNA Primers; D007205:Indium Radioisotopes; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin; D004369:Pentetic Acid