Identification and characterization of the KlCMD1 gene encoding Kluyveromyces lactis calmodulin

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is the major cause of morbidity and death after lung transplantation. Therapy has focused on augmented immunosuppression with a variety of agents. Although transient responses are often achieved, sustained remission has been unusual. The outcome of cytolytic therapy for BOS at our center has been analyzed and is reported. METHODS: Between July 1988 and July 1994, 233 patients underwent lung transplantation at Barnes-Jewish Hospital. Among 207 recipients (88.8%) who survived more than 3 months, 81 recipients (39%) had development of BOS; 48 of these patients underwent 64 courses of treatment with a cytolytic agent (antilymphocyte globulin, antithymocyte globulin, or OKT3 monoclonal antibody). The cases of BOS were retrospectively analyzed to determine the impact of cytolytic therapy. RESULTS: The 4-year survival rate was significantly greater in recipients without BOS than in those with BOS (82.8% vs 46.0%; p < .05). Various clinical factors, including diagnosis, forced expiratory volume in 1 second at onset of BOS, presence or absence of pathologically proven bronchiolitis obliterans, type of transplant operation, cytomegalovirus serologic status, and cytomegalovirus pneumonia, were examined, but no significant predictor of survival after the development of BOS was discerned. The mean decrement in forced expiratory volume in 1 second was significantly reduced by cytolytic therapy (-23.5% +/- 2.3% in the 3 months before therapy vs -9.9% +/- 3.5% in the 3 months after the therapy; p < .002). Nevertheless, the stage of BOS progressed over time in spite of therapy in most cases, and only 4 recipients (4.9%) with BOS remained in a lower BOS stage 2 years after treatment. CONCLUSIONS: Recipients with BOS had a significantly lower survival rate than recipients without BOS. No predictor of survival after the onset of BOS was identified. Although cytolytic therapy decreased the rate of decline in pulmonary function in the 3 months after treatment, the stage of BOS ultimately progressed in most patients.     

Optimization of heterologous protein production in Escherichia coli

Escherichia coli has long been the primary prokaryotic host for the synthesis of heterologous proteins. Recent advances have been made in the expression of complex proteins as soluble, functional molecules, complete with prosthetic groups, disulfide bonds, and quaternary structure. The development of alternative promoter and induction strategies has improved the options available for manipulating the expression conditions, which are frequently critical to soluble yield.     

The mitochondrial genome integrity gene, MGI1, of Kluyveromyces lactis encodes the beta-subunit of F1-ATPase

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the beta-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the "gain-of-function" phenotype found in mgi1 point mutants. The location of Arg435 in the beta-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the beta- and alpha- (MGI2) subunits with the gamma-subunit (MGI5) is likely to be affected by the mutations.     

Optimization of Medium Composition for Production of Recombinant Calf Chymosin from Kluyveromyces lactis in Submerged Fermentation

Response surface methodology was applied to optimize medium components for production of recombinant calf chymosin by Kluyveromyces lactis GG799. The previous data indicated that the most suitable carbon source, nitrogen source, salt and vitamin were glucose, yeast extract, KH2P04 and Ca D-Pantothenate, respectively. The concentration of four media components were optimized by using central composite design of response surface methodology. The optimum medium composition for recombinant calf chymosin production was found to contain glucose 29.84 g·L<'-1>,yeast extract 19.85 g·L<'-1>,KH<'2>PO<'4> 0.1 g·L<'-1> and Ca D-Pantothenate 4.49 mg·L<'-1>.The enzyme activity of recombinant calf chymosin was 722 U·mL<'-1>,which was in an excellent agreement with the predicted value (723 U·mL<'-1>). The production of recombinant calf chymosin from Kluyveromyces lactis GG799 was effectively increased by response surface methodology.     

Expression and purification of a truncated macrophage colony stimulating factor in Kluyveromyces lactis

A truncated human macrophage colony stimulating factor (M-CSF) cDNA encoding amino acid residues from 3 to 149 of the native M-CSF was obtained by using polymerase chain reaction. When inserted into plasmid pCXJ1 and psPHO5 and introduced into Kluyveromyces lactis, it directs the the secretory expression of the biologically active dimeric form of M-CSF. Through a four-step purification protocol, i.e. ammonium sulfate salting out, DEAE-cellulose column chromatography, hydrophobic chromatography on phenyl-sepharose and Mono Q fast protein liquid chromatography, the recombinant truncated M-CSF was purified to homogenerity and show its apparent molecular mass at 21KDa on reduced SDS-PAGE, with a specific activity of 1.21 x 10(7) units/mg protein.     

Fusarium graminearum A 3/5 as a novel host for heterologous protein production

We describe a novel fungal expression system which utilizes the Quorn myco-protein fungus Fusarium graminearum A 3/5. A transformation system was developed for F. graminearum and was used to introduce the coding and regulatory regions of a trypsin gene from Fusarium oxysporum. The protein was efficiently expressed, processed and secreted by the recombinant host strain. In addition, the promoter and terminator of the F. oxysporum trypsin gene have been successfully utilized to drive the expression of a cellulase gene from Scytalidium thermophilum and a lipase gene from Thermomyces lanuginosus in F. graminearum.     

Isolation of a gene encoding a G protein alpha subunit involved in the regulation of cAMP levels in the yeast Kluyveromyces lactis

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0.5 kb (P-1) and 0.8 kb (P-2). Sequencing showed that these two fragments share high homology with genes coding for the G alpha subunits from different sources. Using the P-1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein alpha subunits from Saccharomyces cerevisiae. KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this G alpha subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian G alpha s family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence.     

Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359

In order to further investigate the potential of rifamycins as antiinflammatory drugs, twenty-five semisynthetic rifamycins were tested at concentrations ranging from 10(-9) to 10(-5) M on in vitro human neutrophil functions such as locomotion, superoxide anion production, and degranulation, under different stimulatory conditions. They were also tested as antiproliferative agents on peripheral blood lymphocytes. The present semisynthetic derivatives are in general characterized by their carrying a hydrophilic substituent; they are rifamycin S or rifamycin SV derivatives carrying at C(3) either a carboxyalkyl side-chain or a glycosyl side-chain. Derivatives of the former group displayed inhibitory activities covering the whole range of activities tested, suggesting that the sum of these different effects could support their antiinflammatory activity in vivo. These derivatives, carrying a free carboxyl, are more water soluble than rifamycin SV at physiological pH, and may serve as antiinflammatory drugs for local administration, alternative to rifamycin SV, possibly giving higher efficacy and reduced side effects of pain and tissue swelling.     

The use of heterologous promoters for adeno-associated virus (AAV) protein expression in AAV vector production

OBJECTIVE: There are few nonproprietary papers addressing the mechanical strength of intramedullary nails; none address the characteristics of the proximal and distal ends of these devices. Our objective was to provide such data. DESIGN: Independent testing of eight femoral intramedullary nail systems at the proximal, middle, and distal regions was undertaken to evaluate strength and flexural rigidity (stiffness). METHODS: Each device, usually a reconstruction nail, was forty-two to forty-six centimeters in length. Four or five nails of each available size (range 9 to 13 millimeters in diameter) were tested for each system. The nails were cut into proximal, middle, and distal thirds. Each nail section was loaded to failure using a four-point bend test on a custom fixture (modification of the American Society of Testing Materials standard test). RESULTS: Significant variations (p < 0.05) were found in strength and stiffness between the middle and the proximal or distal aspects of some rods. A significant difference (p < 0.05) was observed when comparing the properties of earlier designs with the properties of more recent designs. Newer rod designs all performed in a similar manner with regard to strength. Strength and rigidity increased with increasing rod diameter in some but not all systems. CONCLUSIONS: Although none of the newer designs appeared to have superior static strength, the individual systems had significant variations in their mechanical properties (bending rigidity), particularly in the proximal and distal sections. It is important that the surgeon become familiar with the individual characteristics of strength and rigidity for the particular devices available and how these might impact fracture healing. Consideration of this information could alter the decision to select one system over another in a complex fracture situation.     

Strategies for optimizing heterologous protein expression in Escherichia coli

The many advantages of Escherichia coli have ensured that it remains a valuable host for the efficient, cost-effective and high-level production of heterologous proteins. Here, we describe the current status of this prokaryotic expression system and focus on strategies designed to maximize the yields of recombinant proteins. Major challenges facing this expression system are also outlined.     

Glucose represses the lactose-galactose regulon in Kluyveromyces lactis through a SNF1 and MIG1- dependent pathway that modulates galactokinase (GAL1) gene expression

Expression of the lactose-galactose regulon in Kluyveromyces lactis is induced by lactose or galactose and repressed by glucose. Some components of the induction and glucose repression pathways have been identified but many remain unknown. We examined the role of the SNF1 (KlSNF1) and MIG1 (KlMIG1) genes in the induction and repression pathways. Our data show that full induction of the regulon requires SNF1; partial induction occurs in a Klsnf1 -deleted strain, indicating that a KlSNF1 -independent pathway(s) also regulates induction. MIG1 is required for full glucose repression of the regulon, but there must be a KlMIG1 -independent repression pathway also. The KlMig1 protein appears to act downstream of the KlSnf1 protein in the glucose repression pathway. Most importantly, the KlSnf1-KIMig repression pathway operates by modulating KlGAL1 expression. Regulating KlGAL1 expression in this manner enables the cell to switch the regulon off in the presence of glucose. Overall, our data show that, while the Snf1 and Mig1 proteins play similar roles in regulating the galactose regulon in Saccharomyces cerevisiae and K.lactis , the way in which these proteins are integrated into the regulatory circuits are unique to each regulon, as is the degree to which each regulon is controlled by the two proteins.     

Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins

The promoter of alcohol dehydrogenase I of the yeast Saccharomyces cerevisiae was studied using Bacillus amyloliquefaciens alpha-amylase as a marker protein. On glucose, activity of the original ADH1 promoter decreases during late exponential, ethanol production growth phase. When 1100 bp (from -414 bp to -1500 bp) of the upstream sequence are deleted, activity increases into the late ethanol consumption phase but the promoter becomes active only after ethanol production growth phase (Ruohonen et al. (1991) Yeast 7, 337-346). We have now restored 300 bp (from -414 bp to -700 bp) upstream of the deletion site and obtained expression from the ADH1 promoter throughout the yeast growth cycle. The restored sequence allowed alpha-amylase expression to start during early exponential growth phase indicating that it is required for activation of the ADH1 promoter during ethanol production growth phase, possibly through glucose induction. On ethanol, all the promoters were active, but the short promoter was temporally activated first, suggesting that the restored sequence is not required for promoter activity during early oxidative growth.     

Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon

The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown. We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose. We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase. The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer. A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels. The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells. A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells. Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose. We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway.     

Functional display of a heterologous protein on the surface of Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A

In this study, we showed that the cell wall anchor of protein A from Staphylococcus aureus is functional in the food-grade organism Lactococcus lactis. A fusion protein composed of the lactococcal Usp45 secretion signal peptide, streptavidin monomer, and the S. aureus protein A anchor became covalently attached to the peptidoglycan when expressed in L. lactis. The streptavidin moiety of the fusion protein was functionally exposed at the cellular surface. L. lactis cells expressing the anchored fusion polypeptide could be specifically immobilized on a biotinylated alkaline phosphatase-coated polystyrene support.     

N-myristylation of the catalytic subunit of cAMP-dependent protein kinase conveys structural stability

Coexpression of the yeast N-myristyltransferase with the murine catalytic subunit of cAMP-dependent protein kinase in prokaryotic cells results in the N-myristylation of the recombinant catalytic subunit. The acylated recombinant catalytic subunit was purified following in vitro holoenzyme formation with a mutant form of the regulatory subunit and compared to the non-myristylated recombinant enzyme and to the mammalian porcine enzyme. All three enzymes are very similar in terms of their kinetic properties and their capacity to reassociate in vitro with the regulatory subunit to form holoenzyme. In contrast, the myristylated recombinant catalytic subunit is significantly more stable to thermal denaturation than the non-myristylated enzyme. Its thermal stability is now comparable to the mammalian enzyme. All three catalytic subunits are significantly more stable to thermal denaturation when they are part of the holoenzyme complex. Each shows an increase in T1/2 of 10 degrees C. This study demonstrates that one function for the myristic acid at the NH2 terminus of the catalytic subunit is to provide structural stability.     

Anaphylactic IgE and IgG1 production for hapten can be enhanced by contrast media

Various side effects have been associated with the clinical use of contrast media. Immunological mechanisms have been proposed but there have been very few experimental studies with animal models. We have attempted to develop murine models to determine whether or not anaphylactic antibodies such as IgE and IgG1 against hapten (DNP) were enhanced with contrast medium (iopamidol) as an adjuvant or if the contrast medium itself produced antibodies of the IgE class. The results showed that anti-hapten IgE and IgG1 production was greatly enhanced with immunogen plus contrast medium. Anti-contrast medium antibodies of the IgE class could not be detected by PCA reactions. The enhancement of IgE and IgG1 production for hapten was associated with IL-4 release by the neutralization test used by monoclonal anti-IL-4 antibodies. This is the first observation to show that contrast media may have a strong adjuvant effect for the production of IgE and IgG1. This murine model demonstrates a possible immunological function of contrast media in vivo.     

An enhanced EBNA1 variant with reduced IR3 domain for long-term episomal maintenance and transgene expression of oriP-based plasmids in human cells

The latent replication of oriP-based plasmids in human cells depends on the viral oriP-binding transactivator EBNA1. In this report, the effect of the internal repeat 3 (IR3 or GlyAla repeat) domain of EBNA1 on long-term maintenance and transgene expression of OriP-based plasmids was examined in dividing human cells. To assess the potential contribution of different isoforms of EBNA1 specifically, the long-term stability of oriP-based plasmids was determined after stable transfection of various CMV-driven EBNA1 genes in EBV-negative human B cells. Episome copy number was quantified using a novel sensitive assay based on human mitochondrial DNA as an internal extrachromosomal control. Using this assay, the standard B95.8-derived EBNA1 was compared with its truncated IR3-deleted, form, as well as a new EBNA1 isoform cloned from Raji. The results of a 6-month study indicate that the isoforms of EBNA1 differ with respect to their efficiency of plasmid maintenance. While the EBNA-1 Raji encoding plasmid was the most stable, the oriP-based vector expressing the truncated EBNA1 (IR3del) gene was lost at a much higher rate than those transducing full size EBNA1s. In parallel, long-term reporter gene expression in various human B cell lines was shown to persist at the highest level with the oriP-based Raji EBNA-1 construct. These results show that the GlyAla domain can positively influence long-term plasmid stability and episomal transgene expression.