Peptide mapping of recombinant proteins.
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Biotechnology↗
Biomedical subjects
H S Lu
Publications and source records attributed to H S Lu.
[Follow-up result of 14 cases of the first metatarsophalangeal joint arthroplasty with titanium total joint prostheses].
An 8-year follow-up study of 14 cases (22 feet) of titanium total 1st metatarsophalangeal joint arthroplasty with two designs (type I and type II) is reported. The follow-up period average was 5 years with a range of 3 to 8 years. The rate of subjective satisfaction was 72.7% (16/22), the incidence of complications 31.8% (7/22), and the rate of revision 27.3% (6/22). The results implicate that titanium total hallux MTP joint arthroplasty is a suitable procedure for osteoarthritis, rheumatoid arthritis and hallux rigidus of the 1st MTP joint, and that type II prosthesis seems better than type I prosthesis. In addition, prosthetic designing, surgical technique and postoperative complications are also discussed in detail.
Adult↗
[A comparative study of therapeutic effects of total versus proximal subtotal gastrectomy in adenocarcinoma of the gastric cardia].
485 patients with adenocarcinoma of the gastric cardia operated in our hospital. 283 cases were treated with total gastrectomy, and 202 cases were treated with proximal subtotal gastrectomy. The age, sex, clinical stage, size of tumour, radicality of surgical resection, lymph node involvement, tumour depth penetration and histological type had no significant difference in the two groups. Analysis of survival rates failed to demonstrate any significant difference between the two types of surgical operation for TNM stage I and II. Total gastrectomy resulted in significantly higher survival rate than proximal subtotal gastrectomy for stage III. The 3- and 5-year survival rate of TNM stage III patients increased by 14.6% and 15.1%, respectively (P < 0.05). Extended total gastrectomy was usually applied for stage IV patients without distant metastasis. If the neoplasm had spread beyond the confines of extended total gastrectomy, in order to eliminate obstruction or bleeding, palliative proximal subtotal gastrectomy or total gastrectomy should be considered.
Adenocarcinoma↗
Molecular cloning and sequence analysis of the monkey and human tissue kallikrein genes.
Cynomolgus monkey renal kallikrein cDNA and genomic human tissue kallikrein gene were cloned. The monkey gene encodes a 257 amino acid (aa) preprokallikrein and exhibits 95% and 92% homology to the human at nucleotide (nt) and aa level, respectively. The monkey gene encodes a 233-aa mature kallikrein versus a 238-aa in human. The human kallikrein gene and urinary kallikrein both contain a Lys-162 instead of the reported Glu-162. Human, monkey and rat renal/pancreatic kallikrein genes evolve with a N-glycosylation containing domain (aa 81-87) which is absent in porcine and is non-glycosylable in mice. Only human kallikrein evolves with an additional Thr-108 and with a N-glycosylation site at aa-141.
Amino Acid Sequence↗
Glycosidase digestion, electrophoresis and chromatographic analysis of recombinant human granulocyte colony-stimulating factor glycoforms produced in Chinese hamster ovary cells.
Recombinant human granulocyte colony stimulating factor (G-CSF) produced in Chinese hamster ovary cells is glycosylated. The carbohydrate compositional analysis indicated that G-CSF molecule contains sialic acid, galactose and galactosamine. By isolation and characterization of the purified glycopeptides obtained from cleavages by Staphylococcal aureus V-8 protease and cyanogen bromide, the O-linked glycosylation site was confirmed to be a Thr residue at position 133. Neuraminidase and O-glycanase digestion followed by sodium dodecyl sulfate polyacrylamide and isoelectric focusing gel electrophoreses distinguished two possible carbohydrate structures attached at Thr-133: structure A, NeuNAc-Gal-beta(1,3)-GalNAc-O-Thr; and structure B, NeuNAc-Gal-beta(1,3)-[NeuNAc]-GalNAc-O-Thr. Different glycoforms, undigested or after glycosidase digestion, can also be separated by ion-exchange or reversed-phase high-performance liquid chromatography. The approach described in this report provides a simple and valuable procedure to characterize glycoprotein structures containing simple carbohydrate moieties.
Amino Acid Sequence↗
Isolation and characterization of three recombinant human granulocyte colony stimulating factor His-->Gln isoforms produced in Escherichia coli.
This report demonstrates that three variant isoforms of recombinant methionyl human granulocyte colony stimulating factor are present in small quantities in the crude preparation solubilized from Escherichia coli inclusion bodies. These isoforms were separated from the main form of the factor during purification and further isolated by a series of cationic exchange chromatographic separations. They exhibit full in vitro biological activity and have slightly lower pI's. Structural characterization of the intact proteins and their isolated peptides by sequence determination and mass spectrometric analysis revealed that they are methionyl granulocyte colony stimulating factors having a His-->Gln replacement at sequence position 53, 157, or 171, respectively. The specific His-->Gln change suggests the occurrence of mistranslation during protein synthesis. These variant forms are chromatographically separable during purification and are not detectable in the final purified form of the factor.
Amino Acid Sequence↗
Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs.
The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.
Chromatography, High Pressure Liquid↗
Detection and quantitation of recombinant granulocyte colony-stimulating factor charge isoforms: comparative analysis by cationic-exchange chromatography, isoelectric focusing gel electrophoresis, and peptide mapping.
Routine quantitation of recombinant human granulocyte colony-stimulating factor charge isoforms in the purified protein product requires development of a reliable analytical method. In this report, isoelectric focusing gel electrophoresis, peptide mapping, and cation-exchange high-performance liquid chromatography are compared and evaluated in the analysis of charge isomers that may be present in the recombinant factor. Due to a lack of sensitivity and reliability, isoelectric focusing gel electrophoresis and peptide mapping are not recommended. However, peptide mapping can distinguish aberrant peptides with differences in charges and provide separation for subsequent structural characterization. By this approach, an N-terminally blocked formylmethionyl species was identified to be the minor charge isoform in the purified preparations of recombinant human granulocyte colony-stimulating factor. In contrast to electrophoresis and peptide mapping, a strong cationic-exchange chromatographic procedure was found to be the most selective, sensitive, and reproducible analytical method. The sensitivity and reliability of the method were evaluated and validated using the formylmethionyl isoform and several deamidated analogs (Gln----Glu) made by site-directed mutagenesis. Recombinant human granulocyte colony-stimulating factor preparations contain a very low to undetectable level of the formylmethionine isoform and have no detectable deamidated isoforms.
Chromatography, High Pressure Liquid↗
Isolation of the neu/HER-2 stimulatory ligand: a 44 kd glycoprotein that induces differentiation of mammary tumor cells.
The neu/HER-2 proto-oncogene (also called erbB-2) encodes a transmembrane glycoprotein related to the epidermal growth factor receptor. We have purified to homogeneity a 44 kd glycoprotein from the medium of ras-transformed cells that stimulates phosphorylation of the Neu protein and retains activity after elution from the polyacrylamide gel. The protein is active at picomolar concentrations and displays a novel N-terminal sequence. Cross-linking experiments with radiolabeled p44 result in specific labeling of Neu, indicating that p44 is a ligand for Neu or a related receptor. The purified protein induces phenotypic differentiation of cultured human breast cancer cells, including altered morphology and synthesis of milk components. This is accompanied by an increase in nuclear area, inhibition of cell growth (probably by cell cycle arrest at the late S or the G2/M phases), and induction of DNA polyploidy. We propose the name Neu differentiation factor (NDF) for p44.
Amino Acid Sequence↗
Post-translational processing of membrane-associated recombinant human stem cell factor expressed in Chinese hamster ovary cells.
This report describes the structure of soluble human stem cell factor isolated from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with stem cell factor (SCF) cDNA, which encodes a leader sequence plus 248 additional amino acids. The 248 amino acids include a hydrophobic transmembrane region at positions 190-212. The isolated material is glycosylated and three bands (apparent M(r) 28,000, M(r) 35,000, and M(r) 40,000) are evident by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After complete deglycosylation, the molecular weight by SDS-polyacrylamide gel electrophoresis is 18,000-19,000. Structural analyses of the intact SCF, the deglycosylated SCF, and a deglycosylated C-terminal peptide were performed by laser desorption, fast atom bombardment, or electrospray mass spectrometry. Pulse-labeling of cells with 35S-labeled Met and Cys resulted in cell-associated glycosylated SCF of M(r) 33,000-45,000 which was converted to M(r) 33,000 by in vitro treatment with glycosidases. During a chase with unlabeled Met and Cys, labeled SCF of M(r) 28,000, M(r) 35,000, and M(r) 40,000 appeared in the medium; it was converted to M(r) 18,000-19,000 by glycosidase treatment. SCF at the surface of the transfected CHO cells could be demonstrated by immunofluorescence. The data obtained indicate that the recombinant human stem cell factor, as isolated, represents proteolytically processed forms containing amino acids 1-165, derived from the initially synthesized membrane-bound form of 248 amino acids. Further characterization indicated that the M(r) 28,000 form is glycosylated at Asn120, the M(r) 35,000 form at Asn120 and Asn65, and the M(r) 40,000 form at Asn120, Asn93, and Asn65. Each form also contains O-linked carbohydrate. The N-linked glycosylation, particularly that at Asn93 and at Asn65, adversely affects in vitro biological activity and receptor binding.
Amino Acid Sequence↗
A study of Chinese knee joint geometry for prosthesis design.
This study for the first time provides the geometric parameters of the knee joint of Chinese, which is indispensible to the design of knee prosthesis used for compatriotic patients. Thirty-five items, including linear, radial and angular measurements, were taken from 105 cadaveric knees and knee X-ray films of 1,100 subjects. The method and calculation for proper correction of the X-ray image magnification and joint cartilage space was established. Correlation was found to exist between the X-ray correction coefficients and the body weight, which formed the basis for individualized correction of X-ray measurements. Statistical analysis revealed that most of the linear and radial measurements were highly related while the angular measurements were independent of others. Principal component analysis showed that the width of femoral condyle might be taken as the leading index in determining the dimension of the knee, and regression functions were established to supply the serial parameters for prosthetic design. Multivariate discriminate functions could aid the selection of knee prosthesis.
Anthropometry↗
Glycosylated and unglycosylated recombinant-derived human stem cell factors are dimeric and have extensive regular secondary structure.
We have recently described the identification, isolation, and characterization of a factor, termed stem cell factor (SCF), which acts on primitive hematopoietic progenitors of the marrow. A soluble form of the factor was isolated from the conditioned medium of a rat cell line (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Tung, W., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201) and rat and human cDNAs have been cloned (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. A., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C.-H., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211). The cDNAs encode amino acids C-terminal to those found in the isolated natural form, including a putative transmembrane domain. This paper describes the structural characterization of soluble forms of recombinant human SCF purified from Escherichia coli (unglycosylated) and from Chinese hamster ovary (CHO) cells (glycosylated). Fluorescence emission spectra indicate that the single Trp residue is present in a hydrophobic environment. Circular dichroism and infrared spectroscopy indicate considerable secondary structure, including both alpha-helix and beta-sheet. Molecular weight determinations by sedimentation equilibrium show that the molecules are dimeric (noncovalently associated), and gel filtration analyses are consistent with this conclusion. The CHO cell-derived SCF is about 30% carbohydrate by weight, with both N-linked and O-linked sugar. The presence or absence of the carbohydrate does not influence the results of the various structural analyses.
Circular Dichroism↗
Amino acid sequence and post-translational modification of stem cell factor isolated from buffalo rat liver cell-conditioned medium.
Stem cell factor (SCF) isolated from culture medium conditioned by Buffalo rat liver cells was subjected to detailed structural analysis. Attempts at direct N-terminal sequencing of the factor indicated that its N terminus is blocked as pyroglutamic acid (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201). The removal of the blocking pyroglutamate by pyroglutamate aminopeptidase allowed sequencing of the polypeptide chain to position 47. Stem cell factor was also digested with CNBr, trypsin, Staphylococcus aureus protease (strain V8), and AspN peptidase to generate different sets of peptides that were then separated by reverse-phase high-performance liquid chromatography and sequenced. Sequence of an internal peptide fragment obtained by cleavage of stem cell factor at a single tryptophanyl peptide bond was also obtained. From these analyses, the complete amino acid sequence could be constructed. The factor as isolated is a single polypeptide of 164 or 165 amino acids. The sequence is confirmatory to a sequence deduced from a cDNA sequence and provides important evidence for C-terminal processing of the polypeptide encoded by cDNA. There are four potential N-linked glycosylation sites. Asn65, Asn72, Asn109, and Asn120. Sequence determination of isolated peptides suggested that Asn120 is glycosylated, Asn65 and Asn109 glycosylated in some molecules but not in others, and Asn72 not glycosylated. Amino acids at three positions, i.e. 142, 143, and 155, could not be detected during sequence analysis. Since the gene sequence codes for Ser, Thr, and Thr at these positions (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. C., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C. W., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211), they could be sites of O-linked carbohydrate attachment. The four cysteines form two intramolecular disulfide bonds, Cys4-Cys89 and Cys43-Cys138.
Amino Acid Sequence↗
Dithyreanitrile: an unusual insect antifeedant from Dithyrea wislizenii.
Dithyreanitrile, a novel sulfur-containing indole alkaloid, was isolated from the seeds of Dithyrea wislizenii (Cruciferae). Dithyreanitrile inhibits feeding of fall armyworm (Spodoptera frugiperda) and European corn borer (Ostrinia nubilalis) larvae. Dithyreanitrile, the first natural product with two sulfur atoms and a nitrile attached to the same carbon, was characterized by X-ray diffraction, spectroscopy, and chemical synthesis.
Alkaloids↗
Inhibition of autoproteolytic activation of interstitial procollagenase by recombinant metalloproteinase inhibitor MI/TIMP-2.
The purification and cloning of a novel metalloproteinase inhibitor (MI or TIMP-2) related to tissue inhibitor of metalloproteinases (TIMP) has been recently described by our laboratory (DeClerck, Y.A., Yean, T. D., Ratzkin, B.J., Lu, H.S., and Langley, K.E. (1989) J. Biol. Chem. 264, 17445-17453; Boone, T.C., Johnson, M.J., DeClerck, Y.A., and Langley, K.E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2800-2804). We have transfected Chinese hamster ovary cells with a vector containing human MI/TIMP-2 cDNA and purified recombinant-derived MI/TIMP-2 (rMI/rTIMP-2) from the conditioned medium of such cells. We have investigated the inhibitory activity of rMI/rTIMP-2 toward rabbit fibroblast interstitial collagenase. The inhibition of activated collagenase by rMI/rTIMP-2 is stoichiometric and consistent with the formation of a 1:1 molar ratio complex. In addition to blocking the activated enzyme, rMI/rTIMP-2 inhibits the conversion of 52-kDa procollagenase to the 42-kDa active enzyme initiated by organomercurials. When plasmin is used as activator, rMI/rTIMP-2 does not inhibit the plasmin-mediated conversion of the 52-kDa proenzyme to the 46-kDa inactive intermediate but blocks further conversion of the 46-kDa intermediate to the 42-kDa active enzyme. The data indicate that rMI/rTIMP-2 blocks the autoproteolytic activation of procollagenase. Also, rMI/rTIMP-2 forms complexes with the 52-kDa procollagenase, the 46-kDa intermediate, and with the 42-kDa activated enzyme which are stable to sodium dodecyl sulfate (SDS), such that the complexes can be visualized by SDS-polyacrylamide gel electrophoresis. It appears that the formation of a SDS-stable complex with procollagenase requires an initial conformational change of the procollagenase brought about by organomercurials or by plasmin cleavage. The data suggest that MI/TIMP-2 may be able to control the extracellular action of certain metalloproteinases not only at the level of the activated enzyme but also at the level of proenzyme activation.
Animals↗
Structural analysis of recombinant proteins prepared by semi-dry electroblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Proteins that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were electroblotted onto polyvinylidene difluoride membranes in procedures to prepare homogeneous recombinant proteins for direct N-terminal sequence analysis. A semi-dry blotting procedure was employed to immobilize protein bands on the membranes for subsequent sequence analysis. This method has been used routinely to evaluate the quality of recombinant proteins, which are present in crude cell extracts produced by different expression systems or under different expression conditions. N-Terminal processing, amino acid misincorporation, as well as the inefficient secretion of recombinant proteins can be detected by direct N-terminal sequence analysis of the purified electroblotted samples. Consequently, time-consuming chromatographic procedures can be eliminated. These procedures are also especially valuable for determining degradation sites of a purified recombinant protein, as well as evaluating multiple gene products expressed by isolated cluster genes.
Amino Acid Sequence↗
Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase.
Pseudomonas mendocina KR1 metabolizes toluene as a carbon source by a previously unknown pathway. The initial step of the pathway is hydroxylation of toluene to form p-cresol by a multicomponent toluene-4-monooxygenase (T4MO) system. The T4MO enzyme system has broad substrate specificity and provides a new opportunity for biodegradation of toxic compounds and bioconversions. Its known activities include conversion of a variety of phenyl compounds into the phenolic derivatives and the complete degradation of trichloroethylene. We have cloned and characterized a gene cluster from KR1 that determines the offO activity. To clone the T4MO genes, KR1 DNA libraries were constructed in Escherichia coli HB101 by using a broad-host-range vector and transferred to a KR1 mutant able to grow on p-cresol but not on toluene. An insert consisting of two SacI fragments of identical size (10.2 kb) was shown to complement the mutant for growth on toluene. One of the SacI fragments, when cloned into the E. coli vector pUC19, was found to direct the synthesis of indigo dye. The indigo-forming property was correlated with the presence of T4MO activity. The T4MO genes were mapped to a 3.6-kb region, and the direction of transcription was determined. DNA sequencing and N-terminal amino acid determination identified a five-gene cluster, tmoABCDE, within this region. Expression of this cluster carrying a single mutation in each gene demonstrated that each of the five genes is essential for T4MO activity. Other evidence presented indicated that none of the tmo genes was involved in the regulation of the tmo gene cluster, in the control of substrate transport for the T4MO system, or in major processing of the products of the tmo genes. It was tentatively concluded that the tmoABCDE genes encode structural polypeptides of the T4MO enzyme system. One of the tmo genes was tentatively identified as a ferredoxin gene.
Amino Acid Sequence↗
The N-terminal sequence of the major erythropoietic factor of an anephric patient is identical to insulin-like growth factor I.
The erythropoietic factors present in an anephric patient with nearly normal hematocrit were isolated from plasma by reversed-phase and gel permeation HPLC. The most active fraction was purified and the analysis of its N-terminal sequence was identical to the published sequence of the human insulin-like growth factor I (IGF I). Recombinant human IGF I had identical elution positions as the isolated erythropoietic factor on reversed-phase HPLC and the same molecular weight on gel permeation HPLC. Furthermore, hrIGF I stimulated erythroid colony formation in human bone marrow cultures as was previously shown for the isolated human erythropoietic factor. These results suggest that IGF I may replace erythropoietin as a stimulator of erythropoiesis in some patients with anemia and renal failure.
Amino Acid Sequence↗