Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein

A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO4-polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, we developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. In this case, a DNA fragment coding for the natural signal sequence, the hTF/2N lobe, and the two stop codons was cloned into the expression vector pNUT, such that the expression of hTF/2N was controlled by the mouse metallothionein promoter and the human growth hormone termination sequences. Baby hamster kidney cells containing this hTF/2N-pNUT plasmid secreted up to 20 mg of recombinant hTF/2N per liter of tissue culture medium. Recombinant hTF/2N was purified from the medium by successive chromatography steps on DEAE-Sephacel, Sephadex G-75, and FPLC on Polyanion SI. The purified protein was characterized by NaDodSO4-PAGE, urea-PAGE, amino-terminal sequence analysis, UV-visible spectroscopy, iron-binding titration, and proton NMR.(ABSTRACT TRUNCATED AT 250 WORDS)     

转基因小鼠乳腺上皮细胞的体外培养及其对催乳素反应的研究

实现转基因生物乳腺反应器对外源蛋白的高效表达是目前生物制药亟待解决的难题。催乳素对泌乳期乳蛋白的合成与分泌具有重要的调控功能。通过转基因小鼠乳腺上皮细胞模型的建立,研究催乳素如何调控乳蛋白的表达,为提高乳腺反应器高效表达外源蛋白提供技术及理论支撑。应用机械破碎及胶原酶消化法,经差速贴壁纯化,成功培养含人转铁蛋白基因的小鼠乳腺上皮细胞,细胞上清液中检测到人转铁蛋白表达。细胞经牛催乳素诱导后人转铁蛋白的表达水平明显升高。利用转基因小鼠乳腺上皮细胞模型,可以进行催乳素和环境因素等对乳腺上皮细胞合成及分泌蛋白能力影响的研究。     

Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice

It is very important to develop an effective, specific, and robust expression cassette that ensures a high level of expression in the mammary glands. In this study, we designed and constructed a series of mammary gland‐specific vectors containing a complex hybrid promoter/enhancer by utilizing promoter sequences from milk proteins (i.e., goat β‐casein, bovine αs1‐casein, or goat β‐lactoglobulin) and cytomegalovirus enhancer sequences; vectors containing a single milk protein promoter served as controls. Chicken β‐globin insulator sequences were also included in some of these vectors. The expression of constructs was analyzed through the generation of transgenic mice. Enzyme‐linked immunosorbent assay (ELISA) analysis revealed that the hybrid promoter/enhancer could drive the expression of recombinant human lactoferrin (rhLF) cDNA at high levels (1.17–8.10 mg/ml) in the milk of transgenic mice, whereas control promoters achieved a very low rhLF expression (7–40 ng/ml). Moreover, the expression of rhLF was not detected in the serum or saliva of any transgenic animal. This result shows that all constructs, driven by the hybrid promoter/enhancer, had high mammary gland‐specific expression pattern. Together, our results suggest that the use of a hybrid promoter/enhancer is a valuable alternative approach for increasing mammary‐specific expression of recombinant hLF in a transgenic mouse model.Mol. Reprod. Dev. 79: 573‐585, 2012. © 2012 Wiley Periodicals, Inc.     

Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.     

Elemental iron does repress transferrin, haemopexin and haemoglobin receptor expression in Haemophilusinfluenzae

The iron repressible nature of Haemophilus influenzae transferrin binding proteins suggests a regulatory role for elemental iron in their expression. The existence of a Haemophilus ferric uptake repressor (Fur) binding motif identified in the promoter region of both tbpA and tbpB further supports this hypothesis. However, a recent study using brain heart infusion growth medium suggested that transferrin binding protein synthesis in H. influenzae was haem- rather than iron-regulated. The present study re-investigates this observation and using a chemically defined medium, we demonstrate that elemental iron haem or protoporphyrin IX can each regulate Haemophilus influenzae transferrin, haemopexin and haemoglobin receptor expression.     

Bovine prolactin promotes the expression of human transferrin in the milk of transgenic mice

The bovine prolactin vector was injected directly into the mammary glands of mice carrying the human transferrin transgene to investigate its effect on the production of human transferrin in milk. The mean levels of human transferrin in two experimental groups were increased by approx. 60% compared with the control group: 1143 ± 196 ng/ml (experimental group 1; two injections) and 1160 ± 189 ng/ml (experimental group 2; three injections) versus 714 ± 75 ng/ml (control group). These findings suggest the potential utility of the prolactin vector for efficient expression of valuable pharmaceutical proteins in transgenic animal mammary glands.     

The Main Regulatory Region in the Murine PSP Gene is a Parotid Gland Enhancer

The murine PSP gene is expressed at a high-level in the parotid glands. To extend the knowledge of parotid gland expression and develop tools for expression of heterologous proteins in this tissue, the regulation of the PSP gene was studied using transgenic mice. High-level parotid gland expression of the PSP gene was indicated to depend on a novel regulatory region situated between –8.0 and –6.5 kb. Together with previous results this indicates that the main regulatory elements in the PSP gene are situated between –8.0 to –3.1 kb. This region was shown to activate a heterologous SV40 early promoter in the parotid glands of transgenic mice, suggesting that the PSP gene is controlled by enhancer sequences. A novel Psp derived 9.7 kb parotid gland expression cassette, Lama IV, carrying all known regulatory regions in the PSP gene was expressed at high-levels in the parotid glands and should prove highly useful for expression of heterologous proteins in the saliva of transgenic mice.     

Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells

Conditional expression of suicide genes in vivo has a wide range of applications in biological research and requires a minimal basal promoter activity in the uninduced state. To reduce basal activity of tetracycline (tc)-inducible target promoters we combined synthetic tet operators in varying numbers with a core promoter derived from the plant viral 35S promoter. An optimized promoter, P_TF, was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines. We linked P_TF to the barnase gene, coding for a highly active RNase from Bacillus amyloliquefaciens. Stable cell clones expressing barnase under control of tTA exerted cell death only after tc withdrawal, correlating with a 10-fold induction of barnase mRNA expression. Directing tTA expression through a neuron-specific enolase promoter (P_NSE) leads to barnase expression and cell death in neuronal cells after tc withdrawal. Taken together, our data demonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene propagation in both cell lines and in transgenic mice are observed.     

Development of Transgenic Mice Expressing Calcitonin as a Beta-lactoglobulin Fusion Protein in Mammary Gland

Expression of foreign proteins in mammalian milk is becoming a widespread strategy for high-level production of recombinant pharmaceuticals, especially those with the most complex post-translational modifications. A milk-specific ovine beta-lactoglobulin (oBLG) promoter was used to drive expression of recombinant calcitonin in mouse milk. A gene construct was generated, consisting of 10.7 kbp of the oBLG gene including its promoter and 3′ flanking region with the calcitonin coding sequences inserted in-frame into the oBLG fifth exon. After microinjection, six founder mice transmitted the transgene to their progeny. RT-PCR confirmed mammary-gland specific expression of recombinant mRNA in most transgenic mice and Western blot analysis confirmed expression of chimeric protein. Calcitonin can thus be expressed under the oBLG promoter and regulatory elements in a mammary-gland specific manner.     

Investigating the interaction of anticancer drug temsirolimus with human transferrin: Molecular docking and spectroscopic approach

In our present study, binding between an important anti renal cancer drug temsirolimus and human transferrin (hTF) was investigated employing spectroscopic and molecular docking approach. In the presence of temsirolimus, hyper chromaticity is observed in hTF in UV spectroscopy suggestive of complex formation between hTF and temsirolimus. Fluorescence spectroscopy revealed the occurrence of quenching in hTF in the presence of temsirolimus implying complex formation taking place between hTF and temsirolimus. Further, the mode of interaction between hTF and temsirolimus was revealed to be static by fluorescence quenching analysis at 3 different temperatures. Binding constant values obtained employing fluorescence spectroscopy depicts strong interaction between hTF and temsirolimus; temsirolimus binds to hTF at 298 K with a binding constant of .32 × 10⁴ M^?1 implying the strength of this interaction. The negative Gibbs free energy obtained through quenching experiments is evident of the fact that the binding is spontaneous. CD spectra of hTF also showed a downward shift in the presence of temsirolimus as compared with free hTF implying complex formation between hTF and temsirolimus. Molecular docking was performed with a view to find out which residues are key players in this interaction. The importance of our study stems from the fact it will provide an insight into binding pattern of commonly administered renal cancer drug with an important protein that plays a pivotal role in many physiological processes.     

Ionic residues of human serum transferrin affect binding to the transferrin receptor and iron release

Efficient delivery of iron is critically dependent on the binding of diferric human serum transferrin (hTF) to its specific receptor (TFR) on the surface of actively dividing cells. Internalization of the complex into an endosome precedes iron removal. The return of hTF to the blood to continue the iron delivery cycle relies on the maintenance of the interaction between apohTF and the TFR after exposure to endosomal pH (≤6.0). Identification of the specific residues accounting for the pH-sensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of 11 charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A, and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF-TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367, and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and release of iron from the hTF-TFR complex.     

Unstable expression of transgene is associated with the methylation of CAG promoter in the offspring from the same litter of homozygous transgenic mice

Transgenic animals have been established for studying gene function, improving animals’ production traits, and providing organ models for the exploration of human diseases. However, the stability of inheritance and transgene expression in transgenic animals has gained extensive attention. The unstable expression of transgene through DNA methyltransferase (DNMT) targeting to the methylation of transgenic DNA such as CAG promoter and Egfp coding region in homozygous transgenic animals is still unknown. In the present study, the offspring from the same litter of homozygous transgenic mice carrying ubiquitously expressed enhanced green fluorescence protein driven by CMV early enhancer/chicken β-actin (CAG) promoter was observed to have unstable expression of transgene Egfp, quantitative PCR, western blot and bisulfite sequencing were conducted to quantify the expressional characteristics and methylation levels in various tissues. The correlation between transgene expression and methylation was analyzed. We have found that transgene expression is dependent on the methylation of CAG promoter, but not Egfp coding region. We have also characterized the correlation between the methylation of CAG promoter and DNMT, and found that only Dnmt3b expression is correlated with the methylation of CAG promoter. In conclusion, Dnmt3b-related methylation of CAG promoter can inhibit the transgene expression and may result in the unstable expression of transgene in the offspring from the same litter of homozygous transgenic mice.     

Expression of the chicken transferrin gene in transgenic mice

The chicken transferrin gene was microinjected into the male pronucleus of fertilized mouse eggs, and the eggs were then implanted into foster mothers. Approximately 15%-30% of the offspring from the injected eggs carried chicken DNA sequences; restriction mapping indicated that multiple copies of the chicken gene had integrated into the genome in a tandem arrangement in most of the mice. Six of the seven mice studied expressed the chicken gene, and in five mice there was a 5 to 10 fold preferential expression of chicken transferrin mRNA in liver compared to that in other tissues. Chicken transferrin was secreted into the serum of five of the mice, where it reached steady state concentrations up to 67 micrograms/ml. Offspring from transgenic parents also expressed the chicken gene; in some cases the expression in offspring was very similar to the parent, but in one line expression in offspring had increased 2 to 4 fold.     

The last intron of the human thrombopoietin gene enhances expression in milk of transgenic mice

Introns can enhance gene expression levels. This effect is known as intron-mediated enhancement, which is different from that of enhancers or promoters. In our previous study, under the control of the cytomegalovirus or goat β-casein promoter, the vector containing intron V-TPOcDNA expressed the highest thrombopoietin (TPO) level, whereas the vector containing TPOgDNA expressed the lowest level. In order to verify whether intron V also improves TPO expression in the milk of transgenic mice, rat whey acidic protein promoter was used as regulatory element to construct mammary gland expression vectors including pTPOWA (containing TPOcDNA), pTPOWB (containing intron V-TPOcDNA), and pTPOWC (containing TPOgDNA). These vectors were transfected into HC-11 cells and the supernatants were analyzed at 48 h. The highest TPO level was found in pTPOWB (795 pg/mL) and the lowest level in pTPOWC (193 pg/mL). Then, corresponding vectors were microinjected into fertilized mice zygotes. Transgenic mice were identified by polymerase chain reaction and Southern blot. Enzyme-linked immunosorbent assay was performed to measure TPO levels in the milk of lactating transgenic mice. The highest and lowest TPO levels were found in transgenic mice carrying intron V-TPOcDNA (2,307 pg/mL) and in transgenic mice carrying TPOgDNA (242 pg/mL), respectively. Thus, intron V remarkably improved TPO expression in transgenic mice.     

Muscle fiber type-predominant promoter activity in lentiviral-mediated transgenic mouse

Variations in gene promoter/enhancer activity in different muscle fiber types after gene transduction was noticed previously, but poorly analyzed. The murine stem cell virus (MSCV) promoter drives strong, stable gene expression in hematopoietic stem cells and several other cells, including cerebellar Purkinje cells, but it has not been studied in muscle. We injected a lentiviral vector carrying an MSCV-EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and observed strong EGFP expression in muscle fibers, primary cultured myoblasts, and myotubes isolated from injected muscles. We also generated lentiviral-mediated transgenic mice carrying the MSCV-EGFP cassette and detected transgene expression in striated muscles. LvMSCV-EGFP transgenic mice showed fiber type-dependent variations in expression: highest in types I and IIA, intermediate in type IID/X, and lowest in type IIB fibers. The soleus and diaphragm muscles, consisting mainly of types I and IIA, are most severely affected in the mdx mouse model of muscular dystrophy. Further analysis of this promoter may have the potential to achieve certain gene expression in severely affected muscles of mdx mice. The Lv-mediated transgenic mouse may prove a useful tool for assessing the enhancer/promoter activities of a variety of different regulatory cassettes.