Expression, purification, and initial characterization of human alanine aminotransferase (ALT) isoenzyme 1 and 2 in High-five insect cells.
Alanine aminotransferase (ALT) is a key enzyme for gluconeogenesis as well as a widely used serum marker for liver injury. We have identified two ALT isoenzymes, ALT1 and ALT2, which are encoded by separate genes. In this study, we described the expression, purification and initial characterization of human ALT1 and ALT2 proteins in High-five insect cells. Human ALT1 and ALT2 were expressed as His-tagged fusion proteins by recombinant baculovirus in insect cells and purified into homogeneity in one step by using immobilized Ni2+-affinity chromatography. Tag-free ALT1 and ALT2 were obtained by cleavage of enterokinase digestion and used for initial characterization of the enzymes. The specific ALT activity of purified fusion or His-tag-removed ALT1 was about 15-fold higher than that of ALT2 and their enzymatic activities decreased quickly at 37 degrees C and -20 degrees C, but were well preserved at -80 degrees C. Nevertheless, the ALT1 and ALT2 activities remained stable in a buffer containing 25% glycerol. The pH profile was similar between hALT1 and hALT2 in that both enzymes remained fully active between pH 6.5 and 8.0. The purified ALT recombinant proteins can not only be used as a reference protein standard for the ALT assay in clinical chemistry, but also will be useful for understanding the biochemical and biological significance of the isoenzymes and for developing ALT isoform-specific assays for clinical or preclinical diagnostic use.
Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Li
L
Liu
Shao
S
Zhong
Rongze
R
Yang
Hong
H
Hu
Daozhan
D
Yu
Dalong
D
Zhu
Zichun
Z
Hua
Alan R
AR
Shuldiner
Richard
R
Goldstein
William J
WJ
Reagan
Da-Wei
DW
Gong
D000410:Alanine Transaminase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D005822:Genetic Vectors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera; D013696:Temperature
D017931:DNA Primers; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D000410:Alanine Transaminase