[Bio panning of human stem cell factor(2) mimetic peptides from phage displayed random peptide library].

OBJECTIVE: To screen human stem cell factor (hSCF) mimetic peptides in vitro with a phage-display random peptide library. METHODS: Phage clones with high hSCF receptor (rc-kit/Ig 1-3)-binding activity was screened from phage-displayed random hepta/dodecapeptide library by phage enzyme-linked immunosorbent assay (ELISA). Phage single DNA was extracted and sequenced. Four kinds of peptide with higher c-Kit/Ig 1-3 binding activity were chosen for synthesis and characterized by using cell proliferation assay with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) method in UT-7 cells. RESULTS: Eleven Ph.D.-C7C clones and eight Ph.D-12 phage clones with high hSCF receptor-binding activity were selected from phage-displayed random hepta/dodecapeptide library, respectively. Sequence analysis showed there were no homologous sequence between hSCF and these screened mimetic peptides except one homologous sequence DPSPHTH found in heptapeptide library. All these four synthesized peptides (CE3, CE16, LE4, and LE20), particularly CE16 and LE20, stimulated UT-7 cell proliferation. CONCLUSION: Four hSCF mimetic peptides were successfully isolated from phage-displayed random peptide library..

Human Flt3 ligand from Pichia pastoris inhibits growth of lymphoma and colon adenocarcinoma in mice.

UNLABELLED: Potent effects of Flt3 ligand (FL) on the development of the immune system have generated much interest in application of FL in cancer immunotherapy. OBJECTIVE: To evaluate the effects of Pichia pastoris secreted rhFL on the growth of mouse EL-4 lymphoma and C26 colon adenocarcinoma injected in syngeneic mice for the first time. METHODS: Mice were placed into one of two treatment groups. 2 x 10(5) EL-4 or C26 cells were injected subcutaneously (SC.) into mice on day 0. Group 1 received subcutaneous PBS injections from Day -7 to Day 14 and group 2 received subcutaneous rhFL injections at 30 microg/day from Day -7 to Day 14. Serial tumor areas were measured. On Day 22, mice from each group were sacrificed, and weight of tumors and spleens were evaluated. Data analysis used Student t tests. RESULTS: Pichia pastoris secreted rhFL resulted in tumor growth delay for both EL-4 lymphoma and C26 colon adenocarcinoma compared with control (P < 0.01). Tumors from rhFL-treated mice were smaller (P < 0.01) than controls while spleens larger (P < 0.01) than controls. Histological examination of tumor sections revealed an obvious increase in regions composed largely of infiltrating cells in the rhFL-treated tumors. Infiltrating cells could be detected in clusters among tumors from mice treated with rhFL whereas these cells were only occasionally detected in sections of control tumors. CONCLUSION: Treatment of rhFL expressed from Pichia pastoris resulted in an antitumor response against EL-4 and C26 tumors injected in syngeneic mice.

[Expression, purification, and characterization of the first three immunoglobulin-like domains of human stem cell factor receptor].

OBJECTIVE: To express the first three immunoglobulin-like domains of human stem cell factor receptor (c-Kit/Ig1-3) in E. coli and HEK293 ET cells and study their binding activity for stem cell factor (SCF). METHODS: In prokaryotic expression system, a double mutant form of c-Kit /Ig1-3 (c-Kit /Ig1-3(DM) was produced by overlap PCR and cloned into pET16b. The recombinant protein was expressed in E. coli BL21 (DE3) and refolded by dilution. In eukaryotic expression system, the gene of c-Kit/Igl13 with eight histidine segments was cloned into pEAK12 and the recombinant plasmid was transfected into HEK293 ET cells. The fusion protein was harvested from the growth medium and purified on Ni-NTA agarose column. The recombinant protein was tested for the receptor binding activity with his-tag pull-down and enzyme-linked immunosorbent binding assay. RESULTS: In E. coli c-Kit /Ig1-3(DM) as produced as an inclusion body and showed low binding activity for SCF after refolding. Two HEK293 ET cell clones that express high levels of c-Kit/Ig1-3 were produced and each clone secreted 2p micro/ml of recombinant protein, whose relative molecular mass was about 58,000. Eukaryotically expressed c-Kit/Ig1-3 had specific binding activity for SCF, and the dissociation constant (Kd) was 9.39 nmol/L. CONCLUSION: c-Kit/Ig1-3 with high receptor binding activity is successfully produced in HEK293 ET cells.

[The transcriptional regulation study on human delta globin gene with CAAT box C-->T point mutation in its promoter].

OBJECTIVE: To study the transcriptional regulation of human delta globin gene with C-->T point mutation at -64 in its promoter. METHODS: Human delta globin genes including wild CAAT box and mutant CAAT box (-64C-->T) were separately cloned into eukaryotic expression vector pcDNA3.1 (-)/Myc-His A which was cut out the strong promoter CMV, transfected MEL cells, and induced by DMSO to express. The transcriptional regulation of human delta globin gene was analysed using semi-quantitative RT-PCR. RESULTS: The expression level of human delta globin gene with mutant CAAT box was 2.2-fold as high as that with wild CAAT box. CONCLUSION: The defective CAAT box of human delta globin gene promoter region may be one of the major reasons for its low expression level.

High-level expression of human stem cell factor fused with erythropoietin mimetic peptide in Escherichia coli.

Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.

Functional expression, purification, and characterization of human Flt3 ligand in the Pichia pastoris system.

Flt3 ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of hematopoietic progenitor and stem cells both in vivo and in vitro. Pichia pastoris transformants secreting high-level rhFL were obtained using 'yeastern blotting' method and the expression level in liquid was about 30 mg/L. rhFL was purified to about 95% purity with overnight dialysis, filtration and an anion-exchange step. Further purification steps employing Sephacryl S-200 and reverse-phase HPLC raised the purity to over 99%. The purified rhFL possessed correct N-terminal amino acid sequence and positive Western blotting bands. SDS-PAGE and mass spectrometry analysis showed molecular weight of rhFL was about 21 and 34 kDa, suggesting that rhFL was glycosylated. The result of capillary electrophoresis showed that its pI is 3.12-4.72. Endo H deglycosylation analysis indicated that there was O-glycosylation besides N-glycosylation in rhFL secreted from P. pastoris. Bioactivity assay showed that the purified rhFL had dose-dependent expansion activity on bone marrow nucleated cells.

[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. CONCLUSION: 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.