Development of recombinant human augmenter of liver regeneration (ALR) for treatment of liver diseases

Abstract

Augmenter of liver regeneration (ALR; Hepatopoietin) is a novel hepatotrophic growth factor that stimulates hepatocyte proliferation by two pathways. Human ALR (hALR) is a protein that consists of 125 amino acids and its gene has been mapped on chromosome 16 next to the Polycystic Kidney Disease gene (PKD1). ALR belongs to Ervlp/ALR protein family, whose members have essential functions in the biogenesis of mitochondria. ALR has also been identified as a FAD-linked sulfhydryl oxidase. As ALR has the ability to stimulate hepatocyte proliferation and protect the liver in a liver specific manner, it is a potential drug for liver diseases. In this project, we have successfully produced several polyhistidine-tagged hALR proteins in Escherichia coli expression system. This approach allows single-step affinity purification. The result demonstrated that addition of 12 histidines molecules to the C-terminus of ALR is the best design by which about 330 mg/L of hALR was obtained from 2-L fermentation culture with 95% purity. The in vitro specificity and biological activity of the modified hALR in promoting liver cell growth were found to be similar to those of hepatocyte growth-promoting factor (pHGF), a commercially available drug in China to treat liver failure. Moreover, the modified hALR protein also possesses the sulfhydryl oxidase activity. Recombinant hALR proteins are not very soluble and readily precipitated in phosphate buffer saline (PBS) or elution buffer. To overcome this problem, the ALR was buffer exchanged into a special solution. The solubility of protein was increased by 50 times from 0.02 mg/ml to 1 mg/ml. Another solution to increase its solubility in MilliQ water was to modify the protein with polyethylene glycol (PEG) to produce pegylated hALR. The pegylated protein was fully active in terms of biological activity and sulfhydryl oxidase activity. These data suggest that modified ALR proteins are effective in hepatocyte regeneration.