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H S Lu

Publications and source records attributed to H S Lu.

At least 37 records · Page 2Linked to original sources
PubMed1996

Refolding and oxidation of recombinant human stem cell factor produced in Escherichia coli.

Oxidative folding of recombinant human stem cell factor (rhSCF) produced in Escherichia coli was investigated in vitro. Folding of denatured and reduced rhSCF involves at least five intermediate forms, I-1 to I-5, detectable by their differences in hydrophobicity using reverse-phase high performance liquid chromatography. Both I-1 and I-2 contain a native-like disulfide bond, Cys4-Cys89 and Cys43-Cys138, respectively, and I-3 forms a mispaired disulfide, Cys43-Cys89. These forms appear to reach steady state equilibrium and are important folding intermediates. I-1 was found to be the prominent intermediate that directly folds into native rhSCF (N); and the thermodynamically less stable I-2 favors rearrangment into I-1. I-3 may serve as an intermediate for disulfide rearrangment between I-1 and I-2. I-4 and I-5, which are disulfide-linked dimers, are in equilibrium with reduced rhSCF and other intermediates and may not play an important role in rhSCF folding. Both trifluoroacetic acid-trapped I-1 and I-2, after isolation by high performance liquid chromatography, proceed with the remaining oxidative folding process after reconstitution. Iodoacetate-trapped I-1 and I-2 contain low alpha-helical content and some tertiary structure, while I-3 and reduced rhSCF have little ordered structure. Gel filtration/light-scattering experiments indicate that reduced rhSCF and iodoacetate-trapped I-1, I-2, and I-3 exist as dimeric forms, indicating that rhSCF dimerization precedes formation of disulfide bonds. I-1, I-2, I-3, and the C43,138A analog lacking Cys43-Cys138 bond are not biologically active or exhibit significantly lower activity. The two disulfide bonds in rhSCF seem to be essential for the molecule to maintain an active conformation required for its receptor binding and biological activities.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of a disulfide-linked human stem cell factor dimer. Biochemical, biophysical, and biological comparison to the noncovalently held dimer.

Distinct from the noncovalently linked recombinant human stem call factor (rhSCF) dimer, we report here the isolation and identification of an SDS-nondissociable dimer produced during folding/oxidation of rhSCF. Experimental evidence using various cleavage strategies and analyses shows that the isolated dimer is composed of two rhSCF monomers covalently linked by four disulfide bonds. The cysteines are paired as in the noncovalently associated dimer except that all pairings are intermolecular rather than intramolecular. Other structural models, involving intertwining of intramolecular disulfide loops, are ruled out. The molecule behaves similarly to the noncovalently associated dimer during ion-exchange or gel permeation chromatography. However, the disulfide-linked dimer exhibits increased hydrophobicity in reverse-phase columns and in the native state does not undergo spontaneous dimer dissociation-association as seen for the noncovalent dimer. Spectroscopic analyses indicate that the disulfide-linked and noncovalently associated rhSCF dimers have grossly similar secondary and tertiary structures. In vitro, the disulfide-linked dimer exhibits approximately 3-fold higher biological activity in supporting growth of a hematopoietic cell line and stimulating hematopoietic cell colony formation from enriched human CD34+ cells. The molecule binds to the rhSCF receptor, Kit, with an efficiency only half that of the noncovalently associated dimer. Formation of intermolecular disulfides in the disulfide-linked dimer with retention of biological activity has implications for the three-dimensional structure of noncovalently held dimer and disulfide-linked dimer.

Amino Acid Sequence↗
PubMed1996

Isolation and structural characterization of recombinant human neu differentiation factor expressed in Escherichia coli.

Recombinant human neu differentiation factor produced in engineered E. coli was isolated and subject to structural characterization. The recombinant molecule can be prepared to apparent purity and is active in stimulating receptor tyrosine autophosphorylation in cultural cells expressing HER2 receptor. The 229 amino-acid polypeptide consists of eight cysteines, of which two cysteines near the N-terminus are disulfide-bonded to form an immunoglobulin-like domain and the remaining six cysteines at the C-terminus cross-link to form an epidermal growth factor-like structure. Detailed chemical characterization of the recombinant molecule by peptide mapping in conjunction with Edman sequencing and mass spectrometry reveals that the bacterially produced recombinant neu differentiation factor preparation is properly folded and contains the correct disulfide structure. The peptide mapping procedure is also useful in identifying abnormal peptides derived from deamidation and oxidation of Asn and Met residues, respectively.

Amino Acid Sequence↗
PubMed1996

In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization.

The effect of oxidation of the methionine residues of Escherichia coli-derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine-containing peptides, using RP-HPLC of Asp-N endoproteinase digests. The initial oxidation rates for Met159, Met-1, Met27, Met36, and Met48 were 0.11 min-1, 0.098 min-1, 0.033 min-1, 0.0063 min-1, and 0.00035 min-1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295-nm Trp peak in the near-UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two- and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of human tissue kallikrein produced in Escherichia coli: biochemical comparison to the enzymatically inactive prokallikrein and methionyl kallikrein.

This report describes bacterial expression, isolation, and characterization of human tissue kallikrein recombinantly produced in Escherichia coli. Successful production of enzymatically active recombinant human kallikrein requires the following processes: expression, solubilization and refolding of prokallikrein, thermolysin activation, and chromatographic separation. All experimental data confirmed that bacterially derived human kallikrein is properly folded and exhibits expected biochemical functions. As confirmed by SDS-PAGE and reverse-phase HPLC, recombinant kallikrein is apparently pure and is devoid of reduced or other partially folded kallikrein forms. Recombinant kallikrein behaves as a monomeric molecule in solution and exhibits full enzymatic activity in hydrolyzing peptide substrates. The molecule can bind to aprotinin to form kallikrein-inhibitor complex at a 1:1 molar ratio. Peptide mapping analysis derived from pepsin digestion of recombinant kallikrein assigned five disulfide bonds which match those of porcine kallikrein predicted from X-ray structure. Peptides containing unpaired cysteines or mispaired disulfide bonds were not detected. Both properly folded prokallikrein and methionyl kallikrein, containing a propeptide and an initiator methionine at their N-termini, respectively, were also produced and isolated. These two molecules are structurally similar to recombinant kallikrein, but are not enzymatically active.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of human tissue prokallikrein and kallikrein isoforms expressed in Chinese hamster ovary cells.

We report here the expression of recombinant human prokallikrein and kallikrein in engineered Chinese hamster ovary cells transfected with a human genomic gene encoding preprokallikrein. At high expression levels, recombinant prokallikrein, an inactive proenzyme form, is predominantly secreted into the culture medium. Upon chromatographic separations, the inactive prokallikrein as well as the mature kallikrein after thermolysin activation of the proenzyme can be prepared to apparent purity. Both prokallikrein and kallikrein can be further separated into two distinct high- and low-molecular-weight isoforms. Kallikrein preparations are fully active in standard kallikrein activity assays such as esterase activity and kinin release from kininogen. Both kallikrein and prokallikrein display multiple molecular forms with differences in both molecular sizes and charges. The structural differences in high- and low-molecular-weight kallikreins or prokallikreins were found to be due to glycosylation, with the high-molecular-weight species glycosylated at three Asn-linked sites and the low-molecular-weight species at two of the three Asn-linked sites. The multiply charged kallikrein isoforms are derived from different numbers of sialic acids attached at the detected Asn-linked carbohydrates. In comparison with kallikrein, prokallikrein appears to show a significant decrease in the magnitude of near uv-circular dichroism bands, suggesting a change in local conformation. This conformational change correlates with the loss of activity in proenzyme due to the presence of propeptide.

Amino Acids↗
PubMed1995

Protein design: a hierarchic approach.

The de novo design of peptides and proteins has recently emerged as an approach for investigating protein structure and function. Designed, helical peptides provide model systems for dissecting and quantifying the multiple interactions that stabilize secondary structure formation. De novo design is also useful for exploring the features that specify the stoichiometry and stability of alpha-helical coiled coils and for defining the requirements for folding into structures that resemble native, functional proteins. The design process often occurs in a series of discrete steps. Such steps reflect the hierarchy of forces required for stabilizing tertiary structures, beginning with hydrophobic forces and adding more specific interactions as required to achieve a unique, functional protein.

Amino Acid Sequence↗
PubMed1995

Mistranslation of a TGA termination codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli.

The mature 109-amino-acid human platelet-derived growth factor B (PDGF-B) peptide is derived by intracellular processing from a 241-amino-acid precursor synthesized in mammalian cells, with removal of 81 N-terminal and 51 C-terminal amino acids. In order to produce directly the mature 109-amino acid PDGF-B peptide as a recombinant protein in Escherichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translation termination codon TGA by in vitro mutagenesis. Expression of this DNA via a plasmid vector in E. coli resulted in production of two distinct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating. Structural characterization employing N- and C-terminal amino acid sequencing and MS analyses indicated that the 15 kDa protein is the expected 109-amino-acid PDGF-B, and that the 19 kDa protein represents a C-terminal extended PDGF-B containing 160 amino acids. Characterization of a unique tryptic peptide derived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon at position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161. Owing to the high rate of translation readthrough of TGA codons in this and occasionally other proteins, it appears that the use of TGA as a translation termination codon for proteins to be expressed in E. coli should be avoided when possible.

Amino Acid Sequence↗
PubMed1995

Detection of neu differentiation factor with a biospecific affinity sensor during chromatography.

A technique using a biospecific affinity sensor, BIAcore, was applied to monitor and determine mammalian cell-derived neu differentiation factor (NDF) in column fractions during chromatography. Specific purified polyclonal antibody against Escherichia coli-derived NDF was chemically bound to the surface of BIAcore sensor chips and the derivatized sensor chips were used to detect the specific binding of NDF. The measurement of NDF at very low levels can be assessed by injecting small volumes of the crude media or column fractions into the BIAcore sensor containing antibody-bound sensor chips. This automated procedure performed under computer programming control allows direct measurement of multiple NDF samples in a short period of time and provides excellent quantitative data, which is not possible using other related methods such as Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stimulatory activity assay on receptor autophosphorylation.

Animals↗
PubMed1995

Post-translational processing of membrane-associated neu differentiation factor proisoforms expressed in mammalian cells.

Expression vectors constructed from human and rat pro-neu differentiation factor (NDF) cDNAs were transfected in Chinese hamster ovary cells for expression of recombinant NDF molecules. Soluble NDF forms were released into culture medium after post-translational processing of the membrane-bound pro-NDF forms. Different human and rat NDF isoforms, after being purified from the culture medium, were subjected to structural and biochemical characterizations. The isolated human and rat NDF isoforms have been proteolytically processed at a specific site at the N terminus, which is different from that observed for the processing of rat or human NDF molecule prepared from natural origins. The processing of each recombinant NDF isoform at its C terminus was heterogeneous but consistently occurred at nearby peptide bonds. Specific N- and C-terminal processing by Chinese hamster ovary cells has resulted in the production of two types (alpha and beta) of recombinant NDFs containing 222-225 amino acid residues. Both human and rat NDF molecules are heavily glycosylated at two of the three potential Asn-linked glycosylation sites and contain O-linked sugars at 11 of the Thr/Ser sites. Glycosylation occurs at a short, Ser/Thr-rich spacer region that connects the N-terminal immunoglobulin homology unit to the epidermal growth factor domain. Cellular phosphorylation assay indicated that these secreted forms contain similar biological activity in receptor tyrosine autophosphorylation of mammary tumor cells.

Amino Acid Sequence↗
PubMed1995

Studies on the structure and function of glycosylated and nonglycosylated neu differentiation factors. Similarities and differences of the alpha and beta isoforms.

Comparative analyses of both glycosylated and nonglycosylated neu differentiation factor (NDF) isoforms revealed significant similarities and differences of their overall structures and functions. Biophysical analyses confirmed that all NDF isoforms are monomeric, but have an extended ellipsoidal shape in solution. All full-length NDFs are similar in secondary and tertiary structures and they contain no alpha-helix but are abundant in beta-strand structures. A small NDF fragment containing only the epidermal growth factor domain is also rich in beta-strand structures, but exhibits tertiary structure different from the long NDF forms. Monoclonal antibodies that selectively recognize epidermal growth factor domains of human NDF-alpha and -beta can specifically bind the respective NDF-alpha and -beta isoforms independent of NDF origins. Western blot analysis and quantitative binding assays further identify that an NDF preparation produced naturally from Rat1-EJ cells contains both alpha and beta isoforms in a 3 to 2 ratio. In receptor-binding competition experiments, human and rat NDF-beta isoforms have higher affinity than NDF-alpha isoforms. NDF-beta isoforms can dramatically enhance the stimulation of DNA synthesis for transfected NIH3T3 cells that overexpress HER-3 and HER-4 receptors, while NDF-alpha isoforms can only stimulate proliferation of HER-4-transfected cells with lower activity. Taken together, NDF-alpha and -beta isoforms share similar gross protein conformations but are biologically distinct.

3T3 Cells↗
PubMed1995

Spontaneous dissociation-association of monomers of the human-stem-cell-factor dimer.

In its native state, recombinant human-stem-cell-factor (SCF) dimer can spontaneously and rapidly undergo hybridization when two different SCF dimer species are incubated together. SCF species differing in molecular charge, e.g., a wild-type SCF form and a variant with Asp at position 10 instead of Asn, were used in the hybridization studies; the original species and newly formed dimer hybrid can be separated and quantified by cationic-exchange h.p.l.c. The hybridization reaches an equilibrium where the ratio of hybrid dimer to each of the original species is 2. Kinetic studies of the initial rate of hybridization enable a rate constant for monomer dissociation to be determined. This rate constant is influenced by pH, temperature and salt concentration. The pH and salt effects suggest that salt bridges between charged amino acids at the monomer-monomer interface may be present. From the temperature effects, the activation energy for monomer dissociation was determined to be 85.6 kJ/mol, which is typical for oligomeric proteins. Heavily glycosylated recombinant SCF from Chinese-hamster ovary cells exchanged equally well with the bacterially derived non-glycosylated SCF, indicating that the attached carbohydrate moieties had no effect on monomer exchange.

Amino Acid Sequence↗
PubMed1995

Soluble kit receptor in human serum.

c-kit encodes the transmembrane receptor tyrosine kinase (Kit) for the recently described ligand stem cell factor (SCF). We have developed an enzyme-linked immunosorbent assay for measuring soluble human Kit and we have used the assay to show high levels of soluble Kit in human serum. The distribution of soluble Kit levels was investigated among 112 normal human serum donors. The mean serum level (+/- SD) was found to be 324 +/- 105 ng/mL with the values falling between 163 ng/mL and 788 ng/mL. No correlation between soluble Kit levels and the sexes or ages of the donors was found. Partial purification using immunoaffinity chromatography allowed us to characterize the soluble Kit from pooled human serum. Antibodies generated to a 497-amino acid recombinant human soluble Kit corresponding to the N-terminal extracellular domain of the receptor recognized the serum-derived soluble Kit by immunoblotting. We found that the serum-derived soluble Kit is glycosylated, with mostly N-linked but also O-linked carbohydrate, and with terminal sialic acid residues. When compared with the recombinant human soluble Kit, the serum-derived material was similar both in size and glycosylation pattern. CNBr cleavage of the isolated serum-derived material followed by amino terminal sequencing confirmed the presence of five peptides expected for the extracellular portion of the Kit molecule. The immunoaffinity purified serum-derived soluble Kit inhibited binding of [125I]SCF to membrane-bound receptor in an in vitro assay. These results indicate that soluble Kit could modulate the activity and functions of SCF in vivo.

Adult↗
PubMed1994

Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl.

A novel megakaryocyte growth and development factor (MGDF) has been identified in aplastic canine plasma, and its cDNAs have been cloned from canine, murine, and human sources. Purified canine MGDF isolated by procedures involving MpI receptor affinity chromatography exists in at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, that share the N-terminal amino acid sequence APP-ACDPRLLNKMLRDSHVLH. Human, dog, and mouse cDNAs for MGDF are highly conserved and encode open reading frames for proteins of 353, 352, and 356 amino acids, respectively, including predicted signal peptides. Canine MGDF and recombinant human MGDF support the development of megakaryocytes from human CD34+ progenitor cell populations in liquid culture and promote the survival of a factor-dependent murine cell line (32D) engineered to express MpI. These biological activities are blocked by the soluble extracellular domain of MpI. These data demonstrate that MGDF is a novel cytokine that regulates megakaryocyte development and is a ligand for the MPI receptor.

Amino Acid Sequence↗
PubMed1994

Properties of variant forms of human stem cell factor recombinantly expressed in Escherichia coli.

The gene for human stem cell factor (SCF) encodes a leader sequence followed by 248 amino acids (Martin et al., 1990, Cell 63, 203). Of these 248 amino acids, the first 189 correspond to an extracellular domain and the remainder correspond to a hydrophobic transmembrane domain plus a cytoplasmic domain. A naturally occurring soluble form, released by proteolytic cleavage after amino acid 165, has been described. An alternatively spliced mRNA, lacking the codons for exon 6, has also been described. Since the amino acids encoded by exon 6 include the proteolytic cleavage site, the form expressed from the alternatively spliced mRNA tends to remain membrane-bound. In the present study, we have begun to explore structure/function relationships within the extracellular domain of SCF. Forms beginning at amino acid 1 (after the leader sequence) and ranging from 127 to 189 at the C-terminus have been recombinantly expressed in Escherichia coli and purified. In addition, forms missing the amino acids encoded by exon 6, forms missing up to 10 amino acids from the N-terminus, and forms with disulfide bond alterations have been expressed and purified. The forms have been characterized structurally, as well as functionally, in quantitative cell proliferation and receptor-binding assays. The results indicate that amino acids 1-141 comprise a structural and functional core and allow conclusions about the necessity of each of the two disulfide bonds for structure and function.

Alternative Splicing↗
PubMed1994

B61 is a ligand for the ECK receptor protein-tyrosine kinase.

A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

Amino Acid Sequence↗
PubMed1994

Peptide map analysis of recombinant human granulocyte colony stimulating factor: elimination of methionine modification and nonspecific cleavages.

Procedures for HPLC peptide map analysis of recombinant human granulocyte colony stimulating factor include reduction and S-carboxymethylation of the denatured protein, as well as protease digestion with Staphylococcus aureus endoproteinase Glu-C followed by reverse-phase liquid chromatographic separations. Under nonoptimized experimental conditions analytical problems including methionine modification during carboxymethylation, as well as generation of large, insoluble fragments and nonspecific cleavages during proteolytic digestion, occurred. These problems have complicated the analysis of peptide digests and affected the performance of HPLC columns. This report describes the elimination of these problems by optimizing peptide mapping procedures. We found that mild reduction and alkylation conditions can prevent methionine modification, while protease digestion in the presence of urea at room temperature alleviates generation of peptides derived from incomplete digestion and nonspecific cleavage by endoproteinase Glu-C. Peptide maps generated using the optimized procedures contain fewer peptide peaks with higher recovery. Elimination of incomplete digestion, which generates fewer larger, insoluble peptides, substantially extends the life of reverse-phase columns. The optimized method reproducibly produced peptide maps suitable for routine analysis.

Amino Acid Sequence↗