Raj Sachdev

Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver-conditioned medium

We have identified a novel growth factor, stem cell factor (SCF), for primitive hematopoietic progenitors based on its activity on bone marrow cells derived from mice treated with 5-fluorouracil. The protein was isolated from the medium conditioned by Buffalo rat liver cells. It is heavily glycosylated, with both N-linked and O-linked carbohydrate. Amino acid sequence following removal of N-terminal pyroglutamate is presented. The protein has potent synergistic activities in semisolid bone marrow cultures in conjunction with colony-stimulating factors. It is also a growth factor for mast cells. In two companion papers, we present the sequences of partial SCF cDNAs, identify SCF as a c-kit ligand, and map the SCF gene to the Sl locus of the mouse.

Primary structure and functional expression of rat and human stem cell factor DNAs.

Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated. Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated. Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells, truncated forms of the rat and human proteins have been expressed in E. coli and mammalian cells and have been shown to possess biological activity. SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures. SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size.

Yeast-derived recombinant human insulin-like growth factor I: production, purification, and structural characterization.

Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted fromSaccharomyces cerevisiae using a yeast α-factor leader to direct secretion. However, approximately 10–20% of the IGF-I was in a monomeric form, the remaining materials being disulfide-linked aggregates. When the purified material was subjected to reverse-phase high-performance liquid chromatography (rp-HPLC), it gave two doublet peaks, I and II. Upon reduction, doublet peaks I and II converged to one doublet peak. This suggests that peaks I and II result from different disulfide structures, and the doublet feature of each peak results from other causes. Different disulfide structures between peaks I and II were also suggested from the near UV circular dichroism of these proteins. Only the peak II was biologically active, indicating that peak II has the correct disulfide structure. Concanavalin A affinity chromatography of the purified peak II doublet showed binding of the subpeak with an earlier rp-HPLC retention time, indicating that it was glycosylated. Sequence analysis of tryptic peptides suggested that Thr29 was the site of glycosylation. Site-directed mutagenesis was used to convert Thr29 to Asn29. This substitution reduced, but did not eliminate IGF-I glycosylation, suggesting additional glycosylation sites. The site of carbohydrate addition was consistent with the model that O-glycosylations occur on hydroxyl amino acids near proline residues in β-turns.

Purification and characterization of soluble forms of human and rat stem cell factor recombinantly expressed by Escherichia coli and by Chinese hamster ovary cells

Stem cell factor (SCF) is a novel, early-acting hematopoietic factor. It was isolated from the medium of a rat cell line in a soluble, processed form (Zsebo et al., 1990, Cell 63, 195). The cloned human and rat genes encode the soluble form plus additional C-terminal amino acids including a hydrophobic transmembrane domain (Martin et al., 1990, Cell 63, 203). We have recombinantly expressed forms of human and rat SCF corresponding to the soluble, processed form in Escherichia coli and in Chinese hamster ovary (CHO) cells. After expression in E. coli, folding and oxidation of the SCF polypeptides are required. The SCFs expressed in CHO cells are secreted into the medium in active state and, like the natural SCF, are glycosylated. Purification of the recombinant SCFs is described. Biological and biochemical characterization includes activity toward responsive human and mouse cell lines, N-terminal amino acid sequences, disulfide bond linkages, and sites of glycosylation.

Growth Kinetics of Escherichia coli and Expression of a Recombinant Protein and Its Isoforms under Heat Shock Conditions

Preinduction culture conditions were found to have significant impact on the expression and post‐translational modification of a recombinant human protein in Escherichia coli under heat shock conditions (30 to 42 °C shift). Higher preinduction growth rates (μg) favored better cell viability, greater cell mass yields, and increased cloned gene expression during induction. Formation of recombinant protein isoforms (those containing Nϵ‐modified lysine residues) exhibited an increasing trend with increasing μg. The different extents of post‐translational modifications were suspected to be linked to the different concentrations of certain heat shock protein chaperones resulting from different μg. In view of the extensive involvement of E.coli heat shock proteins in cellular activities&—;including the synthesis, processing, modification, and degradation of proteins&—;at elevated temperatures, it is believed that μg dictated the cellular resources available for synthesizing the heat shock proteins required for cell survival, which in turn determined the ability of the cells to respond to the heat shock. With a higher μg, both the synthesis of host proteins (as indicated by cell growth and survival) and the cloned gene expression were enhanced. The results demonstrate that there exists an intermediate μg for optimum production of the unmodified foreign protein in a heat shock environment. More importantly, they also illustrate the feasibility of improving the recombinant protein homogeneity in fermentation, thereby facilitating downstream processing.

Phosphate Glass as a Phosphate Source in High Cell Density Escherichia coli Fermentations

Polyphosphate glass (sodium hexametaphosphate, sodium polyphosphate, glassy) was identified and tested as a source of phosphate that supports growth of recombinant Escherichia coli to cell densities over 110 g dry wt/L. Polyphosphate glasses cost no more than sodium and potassium phosphates, are more soluble than any other inorganic phosphate source, and do not form metallophosphate precipitates when mixed with other nutrients in proportions found in fermentation medias. Using a typical fermentation medium, 40% higher cell densities were obtained using polyphosphate glass rather than orthophosphate. Polyphosphate glasses will allow more flexibility in designing and executing high cell density fermentations.

Characterization of two fluorescent tryptophans in recombinant human granulocyte-colony stimulating factor : comparison of native sequence protein and tryptophan-deficient mutants

In order to probe the role of the individual tryptophans of granulocyte-colony stimulating factor (G-CSF) inpH and guanidine HCl-induced fluorescence changes, site-directed mutagenesis was used to generate mutants replacing Trp118, Trp58, or both with phenylalanine. Neither Trp to Phe mutation affected the folding or activity of the recombinant G-CSF, and the material expressed in yeast behaved identically to that expressed inEscherichia coli. All of the G-CSF species responded topH and guanidine HCl in qualitatively the same manner. Trp58 has a fluorescence maximum at 350 nm and is quenched to a greater extent by the addition of guanidine HCl, indicating that it is fully solvent-exposed. Trp118 has a fluorescence maximum at 344 nm, and is less solvent-accessible than Trp58. The analog in which both tryptophans have been replaced with phenylalanine shows only tyrosine fluorescence, with a peak at 304 nm which decreases with increasingpH. The intensity of the tyrosine fluorescence in this analog is much greater than that of the native sequence protein or single tryptophan mutants, indicating that energy transfer is taking place from tyrosine to tryptophan in these molecules. Below neutralpH the tyrosine fluorescence is much greater in the [Phe58]G-CSF than in the [Phe118]G-CSF, indicating that Trp58 might be a more efficient recipient of energy transfer from the tyrosine(s).

Assessing post-induction cellular response in a recombinant E. coli lactose-inducible system by monitoring β-galactosidase levels

SummaryA synthetic lactose-inducible promoter was chosen to study host cell responses to the over-expression of heterologous genes. Fermentations were conducted to compare the effect of induction strategies on the synthesis of β-galactosidase versus the production of recombinant protein. The levels of lactose, IPTG and glucose during induction were manipulated to adjust the utilization of lactose as the inducer and/or the carbon source. In addition, the involvement of the gal operon in lactose metabolism was also explored in order to optimize lactose transport and utilization during induction.