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Effect of Heat and pH Denaturation on the Structure and Conformation of Recombinant Human Hepatic Stimulator Substance

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Abstract

Hepatic stimulator substance (HSS) is a novel liver-specific growth-promoting factor. Although HSS has been successfully crystallized, several properties of this protein have yet to be determined. This study shows that recombinant human HSS (rhHSS) is a dimer with a molecular mass of 31 kDa, The protein is weakly acidic and has an isoelectric point (pI) of 4.50. rhHSS was able to protect hepatoma cells from H2O2-induced apoptosis and to stimulate cell growth. The recombinant protein was thermostable up to 80°C and resistant to changes in pH, as determined by synchronous fluorescence and far-UV circular dichroism (CD). Within the range of pH 4.0–10.0, rhHSS assumed a folded conformation identical to the secondary structure of the original, native protein and a native-like far-UV CD spectrum. Denatured rhHSS could be partly reconstituted with respect to its structure, but not its activity. Thus, rhHSS is a structurally stable protein insensitive to thermal and acid–alkaline denaturation.

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Abbreviations

rhHSS:

recombinant human hepatic stimulator substance

GST:

glutathione S-transferase

CD:

circular dichroism

Trp:

tryptophan

Tyr:

tyrosine

RT:

room temperature

SFS:

synchronous fluorescence spectrum

IPTG:

isopropyl-β-d-thiogalactopyranoside

PAGE:

polyacrylamide gel electrophoresis

BCA:

bicinchoninic acid

PBS:

phosphate-buffered saline

RT-PCR:

reverse-transcriptase polymerase chain reaction

SDS:

sodium dodecyl sulfate

IEF:

isoelectrofocusing

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

fetal bovine serum

PMF:

peptide mass fingerprinting

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide

DMSO:

dimethyl sulfoxide

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Acknowledgments

This work was supported by the National Science Foundation of China (30270503) and Project of Beijing Education Committee. We thank Dr. Li Wen for her careful reading of the manuscript.

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Authors and Affiliations

  1. Department of Cell Biology, Capital Medical University, 10 You An Men Wai Xi Tou Tiao, Beijing, 100069, China

    Zhi-cheng Yang, Lin Yang & Wei An

  2. Municipal Key Laboratory of Beijing for Regulation of Liver Protection and Regeneration, Beijing, China

    Zhi-cheng Yang, Lin Yang, Ying-xia Zhang, He-fen Yu & Wei An

Authors
  1. Zhi-cheng Yang
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  2. Lin Yang
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  3. Ying-xia Zhang
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  4. He-fen Yu
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  5. Wei An
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Correspondence to Wei An.

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Yang, Zc., Yang, L., Zhang, Yx. et al. Effect of Heat and pH Denaturation on the Structure and Conformation of Recombinant Human Hepatic Stimulator Substance. Protein J 26, 303–313 (2007). https://doi.org/10.1007/s10930-007-9072-5

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