Expression, purification, and characterization of cysteine-free mouse P-glycoprotein

Cysteine-free mouse MDR3 P-glycoprotein (Pgp) was constructed by mutagenesis of the nine natural Cys to Ala. The Cys-free protein was expressed in Pichia pastoris and purified. Yield, purity, ATPase activity, K(m)(MgATP), and stimulation of ATPase by verapamil, were similar to wild-type mouse Ppg. Mouse Cys-free Pgp was superior in yield and stability to Cys-free human MDR1 Pgp. Mutants Y1040A and Y1040C were constructed in mouse Cys-free Pgp background. Both showed extremely low ATPase activity, strongly-impaired vanadate-trapping of ADP, and reduced photolabeling by 8-azido-ATP. The results are consistent with the conclusion that Tyr-1040 is located in the MgATP-binding site in NBD2 and is required for correct binding and/or orientation of bound MgATP substrate in Pgp as previously suggested by X-ray structures of other ABC transporters and by sequencing of photolabeled Pgp. The results also support our previous conclusion that both catalytic sites must be intact for normal function in Pgp.     

Chicken deoxyribonuclease: purification,characterization, gene cloning and gene expression

Chicken DNase was purified to apparent homogeneity from the pancreas extract. It showed two isoforms, A and B forms, on cation-exchange chromatography. On SDS-PAGE it was a 30-kDa protein. When analyzed on an electrospray-mass analyzer, form A showed a major mass peak of 30859, and form B, 30882. The enzyme was bound to concanavalin A, indicating its glycoprotein nature. The carbohydrate side chain could be removed by endoglycosidase F. Chicken DNase was activated by metal ions and for half-maximum activation, Mn²⁺ and Mg²⁺ required were 1 mM and 4 mM, respectively. The pH optimum was between 7 and 8 depending on the metal ions used. In the presence of Cu²⁺, it was almost completely inactivated by 0.1 M iodoacetate within 1 min. In the absence of Ca²⁺ at pH 8, chicken DNase resisted to the trypsin or -mercaptoethenol inactivation. When the purified enzyme was subjected to protein sequencing, 93% of the sequence was established. Based on the amino acid sequence, the cDNA of chicken DNase was amplified, cloned and sequenced. The cDNA sequence consisted of 1079 nucleotides in which 67 were of the 5-untranslated region and 166 of the 3 and, in the 5-untranslated region, two types of sequences occurred. The polypeptide chain of 282 amino acids, translated from the open reading frame, was composed of the mature protein of 262 amino acids and a putative signal peptide of 20 amino acids. As compared with mammalian DNases, chicken DNase had an overall 58 ± 61% sequence identity, one less potential N-glycosylation site, and one extra disulfide. The cDNA was cloned into the pET15b expression vector. When induced, active recombinant chicken DNase was expressed in Escherichia coli strain BL21(DE3)pLysS and was present in the insoluble fraction of cell lysates.     

Partial characterization of skeletal myoblast mitogens in mouse crushed muscle extract.

We have utilized a model system to investigate myotrophic factors released by normal adult mouse muscles following a crush injury. We found that saline extracts from gently crushed mouse muscles (CME) contain potent mitogenic activities which act on primary newborn mouse myoblast cultures, as well as on mouse C2 cells, a mouse myoblast cell line. We compared the activity of CME on mouse myoblasts with that of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I), two growth factors known to be mitogenic for primary myoblasts (Allen, Dodson, and Lutein: Exp. Cell. Res., 152:154-160, 1984; DiMario and Strohman: Differentiation, 39:42-49, 1988; Allen and Boxhorn: J. Cell. Physiol., 138:311-315, 1989; Dodson, Allen, and Hossner: Endocrinology, 117:2357-2363, 1985; Florini and Magri: Am. J. Physiol., 256:C701-C711, 1989). We found that CME could act in an additive fashion to saturating doses of bFGF to increase proliferation in myoblast cultures. Additionally, CME acted additively to the combination of saturating amounts of bFGF and IGF-I on both C2 and primary myoblast cultures. We also examined additivity of CME with the combination of saturating doses of bFGF, IGF-I, transferrin (Tf), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), adrenocorticotrophin (ACTH), and macrophage colony-stimulating factor (M-CSF). Our data indicate that CME contains Tf, as well as one or more uncharacterized mitogens for myoblasts which are distinct from Tf, the IGFs, bFGF, EGF, PDGF, M-CSF, and ACTH. These uncharacterized mitogens may act independently of known growth factors to stimulate myoblast proliferation, or may act through modulation of known growth factor activities.     

Dystrophin gene expression in patients with Duchenne muscular dystrophy after myoblast transplantation

Based on originally designed technique of myoblast cultivation and in accordance with the approved by the Russian Ministry of Health "one muscle treatment" protocol of myoblast transplantation to the Duchenne muscular dystrophy patients, the first in Russia clinical trial of this gene correction method was carried out. Immonologically related myoblast cultures (30 to 90 million cells per patient) were injected after all preliminary procedures into tibialis anterior muscles of four boys selected from a group of volunteer recipients (Duchenne muscular dystrophy patients) based on the analysis of a number of surface antigens in donor-recipient pairs. The condition of the patients remained satisfactory during the whole period of post-transplantation follow-up (from 6 months to 1.5 years). Six months after myoblast transplantation the presence of donor DNA or dystrophin synthesis was demonstrated in muscle biopsies of three out of four patients. This result confirms efficacy and safety of the procedure used.     

Various forms of mouse lactoferrins: purification and characterization

This work was conducted to study the microheterogeneity of mouse lactoferrin (LF). Two forms, LF₁ and LF₂, could be purified from uterine luminal fluid by ion-exchange HPLC on a Protein PAK SP 5PW column. Another form, LF₃, was purified from the epididymis homogenate by affinity chromatography on a column of Protein A-Sepharose coupled with the purified LF₂ antibody that was prepared to give no crossreaction with serum albumin. Both LF₁ and LF₂ showed a M_r 74 000 band while LF₃ gave a M_r 70 000 band on reducing SDS–PAGE. All of them were reduced to a M_r 68 000 band after they had been digested with N-glycosidase F. The data from automated Edman degradation confirmed the completely identical 19 amino acid sequences in the N-terminal regions of these three LFs, except the lack of N-terminal Lys–Ala of LF₂/LF₃ in LF₁. LF in tissue homogenates was immunodetected by Western blot procedure using the purified LF₂ antibody. Different amounts of LF with a molecular mass of the 70 000 or 74 000 were distributed in the non-sexual organs such as kidney, spleen, lung, heart and liver and the sexual glands including epididymis, vagina, uterus, ovary and prostate. No LF was detected in stomach, intestine, testis and seminal vesicle.     

Morganella morganii urease: purification, characterization, and isolation of gene sequences

Morganella morganii, a very common cause of catheter-associated bacteriuria, was previously classified with the genus Proteus on the basis of urease production. M. morganii constitutively synthesizes a urease distinct from that of other uropathogens. The enzyme, purified 175-fold by passage through DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 chromatography resins, was found to have a native molecular size of 590 kilodaltons and was composed of three distinct subunits with apparent molecular sizes of 63, 15, and 6 kilodaltons, respectively. Amino-terminal analysis of the subunit polypeptides revealed a high degree of conservation of amino acid sequence between jack bean and Proteus mirabilis ureases. Km for urea equalled 0.8 mM. Antiserum prepared against purified enzyme inhibited activity by 43% at a 1:2 dilution after 1 h of incubation. All urease activity was immunoprecipitated from cytosol by a 1:16 dilution. Antiserum did not precipitate ureases of other species except for one Providencia rettgeri strain but did recognize the large subunits of ureases of Providencia and Proteus species on Western blots (immunoblots). Thirteen urease-positive cosmid clones of Morganella chromosomal DNA shared a 3.5-kilobase (kb) BamHI fragment. Urease gene sequences were localized to a 7.1-kb EcoRI-SalI fragment. Tn5 mutagenesis revealed that between 3.3 and 6.6 kb of DNA were necessary for enzyme activity. A Morganella urease DNA probe did not hybridize with gene sequences of other species tested. Morganella urease antiserum recognized identical subunit polypeptides on Western blots of cytosol from the wild-type strain and Escherichia coli bearing the recombinant clone which corresponded to those seen in denatured urease. Although the wild-type strain and recombinant clone produced equal amounts of urease protein, the clone produced less than 1% of the enzyme activity of the wild-type strain.     

白蚁内切-β—1，4-葡聚糖酶基因的克隆、表达、纯化及酶学性质的研究

提取了台湾家白蚁总RNA并反转录获得eDNA,PCR扩增出白蚁内切葡聚糖酶的基因,并将目的基因分别克隆到大肠杆菌和酿酒酵母载体中,构建了产内切-β-1,4-葡聚糖酶的基因工程菌。由于大肠杆菌会有少量的泄漏表达,而所用的酿酒酵母表达载体是本实验室构建带有INU信号肽的表达载体,故都可采用刚果红平板染色法筛选具有羧甲基纤维素酶（CMCase）活性的重组转化子。利用金属镍亲和层析对大肠杆菌表达的内切-β-1,4-葡聚糖酶进行纯化,CMC酶活检测显示纯化酶的最适温度和最适pH值分别为42℃、6．5;内切-β-1,4-葡聚糖酶的Vmax为0．071mg／mL·min,Km值为80．2712mg／mL。     

Plasma membrane association, purification, and partial characterization of mouse sperm beta 1,4-galactosyltransferase

Gamete recognition in the mouse is mediated, in part, by the binding of sperm surface galactosyltransferase (GalTase) to appropriate substrates in the egg zona pellucida. In this paper, sperm GalTase is shown to be an externally oriented, integral plasma membrane component. GalTase is not peripherally adsorbed to the cell surface, nor is it bound to cell surface glycoside substrates. GalTase can be released from the surface of intact sperm by either mild proteolysis or by detergent under conditions in which the sperm membranes remain intact as judged by double-label indirect immunofluorescence. Detergent-solubilized sperm GalTase has been purified to apparent homogeneity by affinity chromatography and characterized as a beta 1,4-GlcNAc:GalTase by substrate and kinetic analyses. Purified and membrane-bound GalTase both show an unusual thermal inactivation above 39-40 degrees C, whereas other sperm enzyme activities as well as GalTase activity from other cell types are temperature-dependent. Purified sperm GalTase inhibits sperm binding to the egg zona pellucida, consistent with its proposed role during gamete recognition.     

Expression, purification, and characterization of histidine-tagged mouse monoglyceride lipase from baculovirus-infected insect cells

Monoglyceride lipase (MGL) has been produced with the baculovirus-insect cell system. The mouse MGL cDNA was subcloned into a baculovirus transfer vector in frame with a sequence encoding an N-terminal stretch of six histidine residues. Purification to apparent homogeneity was obtained by nickel-chelating chromatography. The final yield was 3 mg of pure enzymatically active MGL per liter of Sf9 cell suspension culture. Analysis by SDS-PAGE and mass spectrometry showed that the recombinant histidine-tagged enzyme had the expected molecular mass. With monoolein as substrate, the specific activity and the apparent K(m) were close to those of rat MGL of adipose tissue.     

Partial purification and characterization of mouse peritoneal exudative macrophage elastase

Mouse peritoneal exudate macrophage elastase can be significantly purified with 60% recovery of the starting activity by affinity chromatography against SDS-treated alpha-elastin covalently linked to agarose beads. The enzyme has an apparent Mr of 26 500 based on SDS-acrylamide gel electrophoresis. Molecular sieving chromatography on Sephadex gel gives a Mr for macrophage elastase of 21 000--28 000. The enzyme is not inhibited by chloromethyl ketone inactivators specific for pancreatic and leukocyte elastase nor by phenylmethylsulfonyl fluoride. Macrophage elastase also does not bind to tritiated diisopropylphosphorofluoridate. The enzyme is inhibited by EDTA and thus appears to be a metallo-protease. Macrophage elastase is resistant to human alpha 1-proteinase inhibitor and to human and mouse alpha 2-macroglobulin. In view of its lack of susceptibility to these endogenous serum proteinase inhibitors, macrophage elastase may play an important role in physiological and pathological remodeling of connective tissues.     

Development of a positive method for male stem cell-mediated gene transfer in mouse and pig

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods with low efficiency of transgenic production. A promising approach for producing transgenic animals by using male stem cells was recently reported by Brinster and Zimmermann (1994: Proc Natl Acad Sci 91:11298-11302) and by Brinster and Avarbock (1994: Proc Natl Acad Sci USA 91:11303-11307). However, in order to apply this technique to producing transgenic animals, some difficulties have to be overcome. These include a satisfactory method for short-term in vitro culture for drug selection after transfection with exogenous DNA, and methods for the use of livestock such as pigs. We developed a new method for transferring foreign DNA into male germ cells. Mice and pigs were treated with busulfan, an alkylating agent, to destroy the developing male germ cells, and liposome/bacterial LacZ gene complexes were introduced into each seminiferous tubule by using a microinjection needle. As a control, lipofectin was dissolved in phosphate-buffered saline at a ratio of 1:1, and then injected into seminiferous tubules. In mice, 8.0–14.8% of seminiferous tubule expressed the introduced LacZ gene, and 7–13% of epididymal spermatozoa were confirmed as having foreign DNA by polymerase chain reaction. The liposome-injected testes were all negative for X-gal staining. These results indicate that some spermatozoa were successfully transformed in their early stages by liposome/DNA complexes. In pigs, foreign DNA was also incorporated efficiently into male germ cells, and 15.3–25.1% of the seminiferous tubules containing germ cells expressed the LacZ gene. The data suggest that these techniques can be used as a powerful tool for producing transgenic livestock. Mol. Reprod. Dev. 46:515–526, 1997. © 1997 Wiley-Liss, Inc.     

Isolation, characterization, and chromosomal location of the mouse enamelysin gene

Mouse enamelysin (Mmp20), a member of the matrix metalloproteinase (MMP) family of extracellular matrix degrading enzymes, shows a high degree of homology with other MMPs, particularly those of the stromelysin/collagenase subfamilies. It is expressed exclusively in ameloblasts and odontoblasts. The mouse enamelysin gene (Mmp20) is made up of 10 exons spanning approximately 65 kb within the MMP gene cluster at the centromeric end of chromosome 9.     

Diabetes mellitus-cell transplantation and gene therapy approaches

Diabetes mellitus affects millions of people in the United States and worldwide. It has become clear over the past decade that the chronic complications of diabetes result from lack of proper blood glucose concentration regulation, and particularly the toxic effects of chronic hyperglycemia on organs and tissues. Pancreas transplants can cure insulin-dependent diabetes mellitus (IDDM). Furthermore, recent advances in pancreatic islet isolation and immunosuppressive regimens have resulted in dramatic improvements in the survival and function of islet allografts. Therefore, islet replacement strategies are becoming increasingly attractive options for patients at risk for severe diabetic complications. A major limitation of these approaches is the small number of organs available for transplantation or islet isolation. Thus, an important next step in developing curative treatments for type I diabetes will be the generation of a replenishable source of glucose-responsive, insulin-secreting cells that can be used for beta cell replacement. This review focuses on approaches to developing robust and widely applicable beta-cell replacement strategies with an emphasis on manipulating beta-cell growth and differentiation by genetic engineering.     

Proline dehydrogenase from Pseudomonas fluorescence: gene cloning, purification, characterization and homology modeling

The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescence was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The 3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K(m) and V(max) values of the P. fluorescence ProDH were estimated to be 35 mM and 116 micromol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30 degrees C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173 and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity. This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescence ProDH.     

Expression, purification and characterization of recombinant mouse PC6B from baculovirus-infected insect cells

We constructed a mouse PC6B truncated mutant and introduced a tag of 6 consecutive histidines at its carboxyl terminus for simple purification. Using the baculovirus expression system and standard enzymatic assay, we obtained recombinant mouse PC6B protein and with enzymatic activity.     

Cloning, characterization, and genomic structure of the mouse Ikbkap gene.

Our laboratory recently reported that mutations in the human I-kappaB kinase-associated protein (IKBKAP) gene are responsible for familial dysautonomia (FD). Interestingly, amino acid substitutions in the IKAP correlate with increased risk for childhood bronchial asthma. Here, we report the cloning and genomic characterization of the mouse Ikbkap gene, the homolog of human IKBKAP. Like its human counterpart, Ikbkap encodes a protein of 1332 amino acids with a molecular weight of approximately 150 kDa. The Ikbkap gene product, Ikap, contains 37 exons that span approximately 51 kb. The protein shows 80% amino acid identity with human IKAP. It shows very high conservation across species and is homologous to the yeast Elp1/Iki3p protein, which is a member of the Elongator complex. The Ikbkap gene maps to chromosome 4 in a region that is syntenic to human chromosome 9q31.3. Because no animal model of FD currently exists, cloning of the mouse Ikbkap gene is an important first step toward creating a mouse model for FD. In addition, cloning of Ikbkap is crucial to the characterization of the putative mammalian Elongator complex.