EXPRESSION AND PURIFICATION OF BIOLOGICALLY ACTIVE RECOMBINANT QUAIL STEM CELL FACTOR IN E. COLI

2000 ◽  
Vol 24 (5) ◽  
pp. 311-317 ◽  
Author(s):  
S D'Costa
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Biologically Active ◽  
Expression And Purification ◽  
E Coli

scholarly journals Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

2021 ◽  
Vol 22 (12) ◽  
pp. 6361
Author(s):  
Eunyoung Lee ◽  
Michelle Novais de Paula ◽  
Sangki Baek ◽  
Huynh Kim Khanh Ta ◽  
Minh Tan Nguyen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ion Exchange ◽  
Stem Cell Factor ◽  
Transmembrane Domain ◽  
Coli Strain ◽  
Exchange Chromatography ◽  
Soluble Form ◽  
Human Stem Cell ◽  
E Coli ◽  
Human Stem Cell Factor

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

Bovine stem cell factor: production of a biologically active protein and mRNA analysis in cattle infected with Trypanosoma congolense

1997 ◽  
Vol 59 (1-2) ◽  
pp. 65-78 ◽  
Author(s):  
Bea Mertens ◽  
Cecilia Muriuki ◽  
Paul Muiya ◽  
Aurelie Andrianarivo ◽  
Simon Mwangi ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Trypanosoma Congolense ◽  
Biologically Active ◽  
Active Protein ◽  
Factor Production ◽  
Mrna Analysis

scholarly journals Differential regulation of stem cell factor mRNA expression in human endothelial cells by bacterial pathogens: an in vitro model of inflammation

Blood ◽  
1994 ◽  
Vol 83 (10) ◽  
pp. 2836-2843 ◽  
Author(s):  
A Koenig ◽  
E Yakisan ◽  
M Reuter ◽  
M Huang ◽  
KW Sykora ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Mrna Expression ◽  
Stem Cell Factor ◽  
Bacterial Pathogens ◽  
Bacterial Strains ◽  
Gram Negative ◽  
E Coli ◽  
Inflammatory Stimuli

Abstract Production of hematopoietic growth factors by endothelial cells plays a pivotal role during inflammatory processes. Stem cell factor (SCF) is known to be expressed constitutively in endothelial cells. To investigate the regulation of this cytokine expression by inflammatory stimuli, we cocultured human umbilical vein endothelial cells (HUVEC) with various gram-negative bacterial strains (Escherichia coli, Yersinia enterocolitica, Chlamydia trachomatis, and Neisseria meningitidis, respectively). Experiments were performed with bacterial concentrations ranging from 10(2) to 10(7) bacteria/mL for 3 hours. SCF- specific mRNA expression was studied using Northern blot analysis. Stimulation with the enteropathogenic bacterial strains Y enterocolitica and E coli resulted in a significant concentration- dependent increase of SCF mRNA expression. Similar results were obtained in coculture experiments with N meningitidis. As shown in experiments with E coli, the accumulation of SCF transcripts was also time-dependent. In contrast, coculture of HUVEC with the intracellular gram-negative strain C trachomatis had no effect on SCF mRNA expression. To elucidate the role of the gram-negative bacterial cell wall components, we stimulated HUVEC with bacterial lipopolysaccharide (LPS). LPS induced a maximal SCF mRNA accumulation within 2 hours followed by decrease of SCF-specific transcripts to the basal level after 24 hours. In addition, we exposed HUVEC to the classical inflammatory cytokine interleukin-1 alpha (IL-1 alpha). Kinetic experiments showed a similar pattern of regulation with an increase of SCF mRNA within 2 hours, persisting up to 12 hours, and a decrease to basal transcription after 24 hours. From these data, we conclude that SCF expression is regulated by inflammatory stimuli, such as IL-1 alpha and bacterial pathogens, suggesting an important role of SCF during inflammation.

scholarly journals A Method for Rapid Screening, Expression, and Purification of Antimicrobial Peptides

2021 ◽  
Vol 9 (9) ◽  
pp. 1858
Author(s):  
Yingli Zhang ◽  
Zhongchen Li ◽  
Li Li ◽  
Ben Rao ◽  
Lixin Ma ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Fusion Proteins ◽  
Expression System ◽  
Inhibition Zone ◽  
Eukaryotic Expression ◽  
Biologically Active ◽  
Rapid Screening ◽  
Expression Vectors ◽  
Expression And Purification ◽  
E Coli

In this study, a method for the rapid screening, expression and purification of antimicrobial peptides (AMPs) was developed. AMP genes were fused to a heat-resistant CL7 tag using the SLOPE method, and cloned into Escherichia coli and Pichia pastoris expression vectors. Twenty E. coli and ten P. pastoris expression vectors were constructed. Expression supernatants were heated, heteroproteins were removed, and fusion proteins were purified by nickel affinity (Ni-NTA) chromatography. Fusion proteins were digested on the column using human rhinovirus (HRV) 3C protease, and AMPs were released and further purified. Five AMPs (1, 2, 6, 13, 16) were purified using the E. coli expression system, and one AMP (13) was purified using the P. pastoris expression system. Inhibition zone and minimum inhibitory concentration (MIC) tests confirmed that one P. pastoris¬-derived and two E. coli-derived AMPs have the inhibition activity. The MIC of AMP 13 and 16 from E. coli was 24.2 μM, and the MIC of AMP 13 from P. pastoris was 8.1 μM. The combination of prokaryotic and eukaryotic expression systems expands the universality of the developed method, facilitating screening of a large number of biologically active AMPs, establishing an AMP library, and producing AMPs by industrialised biological methods.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

Stem Cell Factor

2002 ◽  
Vol 14 (2) ◽  
pp. 0052-0059 ◽  
Author(s):  
Susan Smith ◽  
Adrian Piliponsky ◽  
Mor-Li Hartman ◽  
Francesca Levi-Schaffer
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor
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