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H Lubon

Publications and source records attributed to H Lubon.

At least 19 recordsLinked to original sources
PubMed2001

Recombinant human protein C expression in the milk of transgenic pigs and the effect on endogenous milk immunoglobulin and transferrin levels.

Colostrum and milk are natural vehicles for acquiring passive immunity and are valuable tools for decreasing neonatant mortality from diarrheal disease. The effects of recombinant human protein C (rhPC) expression levels on endogenous immunoglobulin and transferrin content of the milk of different lineages of transgenic pigs were studied. The levels of rhPC in the milk ranged from 40 to 1200 microg/ml. Transgenic pigs with rhPC expression levels less than 500 microg/ml had no significant differences in milk protein composition with respect to nontransgenic pigs. A line of transgenic pigs having rhPC expression levels of 960-1200 microg/ml had two- to three-fold higher IgG, IgM, and secretory IgA concentrations compared to other transgenic and nontransgenic pig groups (P < 0.05), and four- to five-fold higher transferrin levels than nontransgenic pigs (P < 0.05). Changes in milk protein composition were not associated with mastitis or other pathologic disruption of epithelial cell junctions as indicated by normal casein and albumin levels in milk. Since IgG, IgM, secretory IgA, and transferrin are transported into the milk by transcytosis, higher levels of these proteins indicate that transcyctosis in the mammary epithelial cell was likely upregulated in pigs having high rhPC expression levels. This study is the first that shows a statistically significant example that mammary tissue specific expression of a heterologous protein can enhance endogenous phenotypic characteristics of milk.

Albumins↗
PubMed2000

DNA preparation method can influence outcome of transgenic animal experiments.

In our continuing quest to improve the efficiency of producing transgenic animals, we have compared the influence of two transgene purification techniques on the efficiency of creating transgenic sheep and mice. Three hundred eighty-seven sheep zygotes and 2,737 mouse zygotes were microinjected with one of four transgenes. Transgenes were isolated from plasmid sequences either by agarose gel electrophoresis followed by gel extraction or by a single step sodium chloride gradient fractionation technique. Four transgenic sheep and 61 transgenic mice were produced. Both sheep and mice embryos responded similarly to transgene preparation methods. Overall, pregnancy rate was higher for recipients that received embryos injected with NaCl purified DNA (mean +/- SEM: 64 +/- 7% vs. 38 +/- 7%). Furthermore, offspring per zygote transferred (NaCl, 22 +/- 3% vs. Gel, 12 +/- 3%) and transgenics born per zygote transferred (NaCl, 3.9 +/- 0.6% vs. Gel, 1.5 +/- 0.6%) were higher when the NaCl purified DNA was used. However, the proportion of offspring born that were identified as transgenic did not differ between transgene purification methods. Transgenes responded differently to methods of preparation. One of the four genes yielded a significantly higher proportion of transgenics when the transgene was prepared by NaCl purification. These data suggest that on average the NaCl gradient purification technique results in a higher embryo survival rate to term for both sheep and mice, but the technique has no influence on rate of transgene integration.

Animals↗
PubMed1999

Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland.

The mammary gland of transgenic livestock can be used as a bioreactor for producing complex therapeutic proteins. However, the capacity for making a given post-translational modification upon any given polypeptide is uncertain. For example, the efficiency of gamma-carboxylation of glutamic acid in the amino terminal regions of recombinant human protein C (rhPC) and recombinant human Factor IX (rhFIX) is different at similar expression levels. At an expression level of about 200 microg/ml in the milk of transgenic pigs, rhFIX is highly gamma-carboxylated as indicated by pro-coagulant activity and amino acid sequencing. However, only about 20-35% of rhPC has a native, gamma-carboxyglutamic acid-dependent conformation and anti-coagulant activity. Thus, this work provides an example of apparent differences in substrate specificity between two homologous proteins to the endogenous carboxylase of porcine mammary epithelium which leads to varying degrees of post-translational modification.

Amino Acid Sequence↗
PubMed1998

Transgenic animal bioreactors in biotechnology and production of blood proteins.

The regulatory elements of genes used to target the tissue-specific expression of heterologous human proteins have been studied in vitro and in transgenic mice. Hybrid genes exhibiting the desired performance have been introduced into large animals. Complex proteins like protein C, factor IX, factor VIII, fibrinogen and hemoglobin, in addition to simpler proteins like alpha 1-antitrypsin, antithrombin III, albumin and tissue plasminogen activator have been produced in transgenic livestock. The amount of functional protein secreted when the transgene is expressed at high levels may be limited by the required posttranslational modifications in host tissues. This can be overcome by engineering the transgenic bioreactor to express the appropriate modifying enzymes. Genetically engineered livestock are thus rapidly becoming a choice for the production of recombinant human blood proteins.

Amino Acid Sequence↗
PubMed1997

Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C.

The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100-1800 micrograms ml-1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100-400 micrograms ml-1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein.

Animals↗
PubMed1997

Transgenic pigs produce functional human factor VIII in milk.

Deficiency or abnormality of coagulation factor VIII (FVIII) causes a bleeding disorder called hemophilia A. Treatment involves FVIII concentrates prepared from pooled human plasma or recombinant FVIII (rFVIII) prepared from mammalian cell culture. The cost of highly purified FVIII or rFVIII is a major factor in hemophilia therapy and restricts prophylaxis. We have sought to generate a new source of rFVIII by targeting expression of the human FVIII cDNA to the mammary gland of transgenic pigs using the regulatory sequences of the mouse whey acidic protein gene. The identity of processed heterodimeric rFVIII was confirmed using specific antibodies, by thrombin digestion and activity assays. The secretion of as much as 2.7 micrograms/ml of rFVIII in milk was over tenfold higher than in normal plasma. Up to 0.62 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.

Animals↗
PubMed1996

Rate limitations in posttranslational processing by the mammary gland of transgenic animals.

Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs. Human protein C (hPC) is a complex plasma protein containing nine gamma-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM. Gamma carboxylation is a vitamin K-dependent posttranslational modification. The effect of rhPC synthesis rate on the extent of gamma-carboxylation of glutamic acid was studied. We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression. Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes. Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 +/- 4 micrograms rhPC/ml of milk and 895 +/- 21 micrograms rhPC/ml of milk, respectively. Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 micrograms rhPC/ml of milk and 800 to 2000 micrograms rhPC/ml of milk, respectively. A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC. Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC. Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response. This study suggests that a rate limitation in gamma-carboxylation by the mammary gland occurs at expression levels about > 20 micrograms/ml in mice and > 500 micrograms/ml in pigs.

Amino Acid Sequence↗
PubMed1996

Activation of recombinant human protein C.

We have produced recombinant human Protein C (rHPC) in the milk of transgenic swine. After purification, we have analyzed the interaction of teh zymogen with Protac, thrombin/thrombomodulin and thrombin alone. The amidolytic and anticoagulant activities of rAPC after Protac activation were approximately 80% those of its human plasma counterpart. Upon the excision of the activation peptide by thrombin/thrombomodulin complex, both the natural and recombinant activation products had similar enzymatic and biological activities. This observation can be attributed to the difference in the mechanism of action between the two activators and structural differences between HPC and rHPC.

Animals↗
PubMed1995

Thermal stability and domain-domain interactions in natural and recombinant protein C.

Scanning microcalorimetry and spectrofluorimetry were applied to a study of the thermal stability and interaction of the modules within natural human protein C (PC) and recombinant protein C (rPC), a potential therapeutic anticoagulant expressed in transgenic pigs. Upon heating in the presence of 2 mM EDTA, pH 8.5, each protein exhibited a similar heat absorption peak with a Tm of approximately 62 degrees C corresponding to the melting of the serine protease (SP) module. Deconvolution of this peak indicated that the SP module consists of two domains that unfold independently. At pH below 3.8, a second peak appeared at extremely high temperature corresponding to the unfolding of the two interacting epidermal growth factor-like (EGF) domains. This second peak occurred at a temperature about 20 degrees C lower in rPC than in PC indicating that the EGF domains in the recombinant protein are less stable. The isolated 6-kDa gamma-carboxyglutamic acid-rich (Gla) fragment as well as a 25-kDa Gla-(EGF)2 fragment both exhibited a sigmoidal fluorescence-detected denaturation transition in the same temperature region as the SP domains, but only in the presence of Ca2+. In 2 mM Ca2+, the first heat absorption peak in both intact proteins became biphasic, indicating Ca(2+)-induced structural changes. By contrast, Ca2+ had very little effect on the melting of Gla-domain-less protein C. This indicates that not Ca2+ itself but the Ca(2+)-loaded Gla domain is responsible for conformational changes in the SP domain of the parent protein. Detailed analysis of the shape of the endotherms obtained in Ca2+ and EDTA suggests that Ca2+ induces compact structure in the Gla domain which appears to interact strongly with the SP domain(s) of protein C.

Calcium↗
PubMed1995

Proteolytic processing of human protein C in swine mammary gland.

Maturation of human Protein C (HPC) precursor to a zymogen in the liver requires endoproteolytic cleavages after a basic dipeptide, Lys-2-Arg-1 in the propeptide and Lys156-Arg157 connecting the light and heavy chain. Recombinant human Protein C (rHPC) was expressed in the mammary gland of transgenic swine and its proteolytic processing was monitored. We found that about 10-20% of rHPC purified from the milk still retained the propeptide and 30-40% was in the single-chain form, indicating inefficient proteolytic cleavage. This demonstrates that endoprotease(s) of the swine mammary epithelial cells do not process fully the precursor of heterologous protein. rHPC was fractionated by anion exchange chromatography and polypeptides with novel N-termini at positions -1,152 and 157 were detected in addition to the known N-termini at residues -24, +1, and 158. Since rHPC was found to be stable both in milk and after purification, it is possible that these new cleavages on the amino side of arginine at dipeptide sites Lys-2-Arg-1, Lys151-Arg152, and Lys156-Arg157 could have occurred in the mammary gland. Thus, our results suggest that a portion of HPC precursor was proteolytically processed in swine mammary gland differently than those in other expression systems.

Amino Acid Sequence↗
PubMed1994

The porcine mammary gland as a bioreactor for complex proteins.

The similar biological activity of rhPC and hPC indicates that porcine mammary gland can perform many of the processing reactions necessary for recombinant synthesis of complex human proteins and produce them at levels suitable for industrial bioreactor applications. The health of the transgenic pigs appeared unaffected by the expression of high levels of the heterologous protein. We suggest that one of the advantages of using the mammary gland as a bioreactor appears to be the high cell density relative to that of cell culture.

Animals↗
PubMed1994

Regulation of human protein C gene expression by the mouse WAP promoter.

A 4.1 kb mouse whey acidic protein (mWAP) promoter was cloned from a C57BL/6 cosmid library. The tissue-specific and developmental pattern of expression of a hybrid gene comprised of the mWAP promoter fragment and the human protein C (HPC) gene was analysed in transgenic mice. The corresponding RNA was detected mainly in the mammary gland, with 'leakage' of expression in the salivary gland and kidney. The developmental pattern of transgene expression differed from that of the endogenous WAP gene. In particular, recombinant HPC (rHPC) transcripts were detected earlier in pregnancy than WAP RNA, with no significant increase during lactation. This indicates that regulatory elements responsible for developmental regulation are located outside the 4.1 kb mWAP gene promoter fragment, or if present, may be subject to position effects. Precocious expression of the transgene did not compromise the health or nursing abilities of transgenic females. Expression of rHPC affected the appearance of the mammary alveoli and alveolar epithelial cells in lactating transgenic mice. The alveoli were less distended and alveolar epithelial cells appeared cuboidal with centrally positioned nuclei. We suggest that the inefficient intracellular processing of rHPC can alter the histological appearance of alveolar epithelial cells in the transgenic mammary gland.

Animals↗
PubMed1994

Inefficient processing of human protein C in the mouse mammary gland.

Vitamin K-dependent plasma protein, human Protein C (HPC) has been expressed in transgenic mice, using a 4.2 kb mouse whey acidic protein (WAP) promoter, 9.0 kb HPC gene and 0.4 kb 3' flanking sequences. Expression was mammary gland-specific and the recombinant human Protein C (rHPC) was detected in milk at concentrations of 0.1 to 0.7 mg ml-1. SDS-PAGE revealed that the single, heavy and light chains of rHPC migrated with increased electrophoretic mobility, as compared to HPC. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation. The substantial increase observed in the amount of single chain protein, as well as the presence of the propeptide attached to 20-30% of rHPC, suggest that mouse mammary epithelial cells are not capable of efficient proteolytic processing of rHPC. The Km of purified rHPC for the S-2366 synthetic substrate was similar to that of plasma-derived HPC, while the specific activity was about 42-77%. Amino acid sequence analyses and low anticoagulant activity of purified rHPC suggest that gamma-carboxylation of rHPC is insufficient. These results show that proteolytic processing and gamma-carboxylation can be limiting events in the overexpression of fully biologically active rHPC in the mouse mammary gland.

Amino Acid Sequence↗
PubMed1990

Interactions between cellular regulatory proteins and a unique sequence region in the human cytomegalovirus major immediate-early promoter.

Transcription from the human cytomegalovirus major immediate-early promoter is dependent on host-cell regulatory proteins. The interactions between cellular nuclear proteins and a unique sequence located from nucleotide position -660 to -540 was investigated. The unique region presents a defined target for multiple distinct DNA-binding proteins which appear, in part, to have overlapping binding sites. A minimum of five sequence-specific DNA-binding activities that interact with sequences between -632 and -602, -602 and -557, -602 and -590, -563 and -540, and -602 and -582 were detected. Evidence is presented to suggest that the -632 to -602 site, a previously characterized nuclear factor 1 binding site, does not bind NF1 but strongly interacts with a distinct cellular factor. The binding of cellular proteins to the unique sequence region was shown to be important in directing transcription from the major immediate-early promoter.

Antigens, Viral↗