Abstract
Human stem cell factor initiates a diverse array of cellular responses, including hematopoiesis, cell proliferation, differentiation, migration and survival. To explore the relationship between its structure and function, we produced recombinant soluble human stem cell factor1–165 (wild type) and human stem cell factor1–141 (C-terminal truncated) in a yeast expression system and compared their biological activities and thermal stabilities. The biological activity of the two proteins was measured as a function of TF-1 cell viability and effects on downstream signaling targets after incubation. We found that these proteins enhanced cell viability and downstream signaling to a similar extent, in a dose-dependent manner. The biological activity of recombinant human stem cell factor1–165 was significantly greater than that of recombinant human stem cell factor1–141 after heating the proteins (100 ng/mL) at 25–110°C for 10 minutes (P<0.05 for all temperatures). In addition, circular dichroism spectral analysis indicated that β-sheet structures were altered in recombinant human stem cell factor1–141 but not recombinant human stem cell factor1–165 after heating at 90°C for 15 or 30 min. Molecular modeling and limited proteolytic digestion were also used to compare the thermo stability between human stem cell factor1–165 and human stem cell factor1–141. Together, these data indicate that stem cell factor1–165 is more thermostable than stem cell factor1–141.
Highlights
Human stem cell factor is a glycoprotein cytokine that induces Kit activity
Several clones with the Mut+ phenotype that expressed the maximal levels of hSCF1–165 or hSCF1–141 were chosen in small-scale expression and stored in Yeast Extract-Pepttone-Dextrose (YPD) containing 15% glycerol for further scale-up cultures
The plasmids pPICZaC-rhSCF1–141 and pPICZaC-rhSCF1–165 were integrated into the alcohol oxidase promoter (AOX I) locus of the P. pastoris chromosome
Summary
The effects of SCF are exerted through at least 4 intracellular pathways and involve Src family members, phosphatidylinositol-3-kinase, the Janus family of protein tyrosine kinases (Jak), and the Ras-Raf-mitogen activated protein (MAP) kinase cascade These pathways mediate a number of cellular processes, including gene transcription, proliferation, differentiation, survival, metabolic homeostasis, melanin pigmentation, development, and cell migration [1,2,3,4,5,6,7,8,9,10]. SCF is expressed as two different isoforms composed of 220 and 248 amino acids These membrane-associated proteins are generated via alternative splicing of the same RNA transcript [11], the latter of which includes a proteolytic cleavage site in exon six. Each SCF monomer contains two intra-chain disulfide bridges
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