[Expression of human HSF in E. coli and its effects on mobilization of hematopoietic stem cells in rhesus monkeys].
OBJECTIVE
To study the expression of hHSF in E. coli and its effect on the mobilization of hematopoietic stem/progenitor cells.


METHODS
The hHSF gene was obtained by overlapping PCR and cloned into the vector pET30a to yield pET30a-hHSF, which was transformed into E. coli BL21(DE3) and expressed with IPTG induction. Subsequently, rhHSF was purified by gel filtration and cation exchange chromatography and subjected to refolding. Molecular weight of hHSF was measured by MALDI-TOF Mass Spectroscopy. The N terminal amino acid sequence rhHSF was determined by protein sequencing. rhHSF was profiled in rhesus monkey for mobilization of peripheral blood stem cells. Eight rhesus monkeys were equally divided into two groups. The first group was administered single subcutaneous injection of 500 microg/kg hHSF, while the other one was administered 10 microg.kg(-1).d(-1) G-CSF for 4 days followed by a single subcutaneous injection of 500 microg/kg rhHSF.


RESULTS
The sequence coding hHSF was confirmed by sequencing and the induced-expression level was about 30% of total cell proteins. The purity of target protein was over 95%. The sequence of N terminal 10 amino acids and the amino acid composition were consistent with the theoretical parameters; molecular weight of rhHSF was 7540. The peripheral CD34(+) cells, CFU-GM yields, and neutrophils peaked at 3 h (16.3-folds increase compared with baseline), 1 h (1.9-folds increase) and 45 min (4.4-folds increase) respectively after the single injection of rhHSF. The addition of rhHSF after the last dose of G-CSF boosted these levels to 25.8-folds, 8.7-folds and 8.3-folds respectively.


CONCLUSIONS
hHSF is highly expressed in E. coli and rapidly mobilizes the hematopoietic stem/progenitor cells and neutrophils in rhesus monkeys. hHSF shows distinct synergistic effect with G-CSF.
Qi
Chun-mei
CM
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS & PUMC, Beijing 100005, China.
Yang
Ke-gong
KG
Chen
Song-sen
SS
Zhang
Yan-li
YL
Deng
Yan-chun
YC
Su
Lin
L
Wang
Ya-Dong
YD
Luo
Si
S
Liu
Chang-zheng
CZ
Luo
Qing-liang
QL
Xiong
Guo-Lin
GL
D000818:Animals; D054426:Chemokine CXCL2 / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D008253:Macaca mulatta; D008297:Male; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology*; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C499706:CXCL2 protein, human; D054426:Chemokine CXCL2; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor