Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris

High-level expression and efficient assembly of hepatitis B surface Antigen (HBsAg) has been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under control of the AOX1 promoter. To fully utilize the expression potential of the P. pastoris expression system, we investigated the influence of gene copy number on the expression of HBsAg in this yeast. A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. Using this strategy, constructs containing up to a maximum of eight direct repeats of the HBsAg-expressing cassettes could be created. These expression cassettes were targeted for integration into the genome of the host strain GS115 with simultaneous elimination of the resident AOX1 gene. Deletion of the AOX1 gene was intended to create Mut(s) (methanol utilization slow) transformants that are known to have an increased ability to generate HBsAg in particulate form. A systematic investigation of the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the steady-state levels of the HBsAg-specific mRNA, which in turn is closely paralleled by a corresponding increase in the total levels of the HBsAg protein. Virtually all the recombinant protein in the soluble fraction was present in the particulate form based on particle-specific ELISA and sedimentation behavior. Further, our studies also revealed the continued physical and functional integrity of the HBsAg-expressing cassettes during the course of an extended induction phase spanning 6 days.     

Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter

High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter. However, the time taken to reach peak product concentration is usually very long ( approximately 240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in secretion of approximately 20 nm HBsAg particles as evidenced by electron microscopy. However, the levels of secreted HBsAg particles were very low, presumably due to the inherent hydrophobicity of the HBsAg molecule and the consequent propensity for membrane association. Our studies show that secretion is not a good strategy for expression of HBsAg in P. pastoris. The data also suggests that intracellular production of HBsAg under the GAP promoter using multicopy expression cassettes can indeed serve as an effective alternative to the AOX1 promoter. Further, the GAP promoter based system obviates the need to use and extensively monitor methanol during recombinant antigen production. Finally, this constitutive system has the potential for continuous culture wherein several batches of recombinant protein-containing biomass can be harvested from a single initial fermentation.     

Endocardial activation during ventricular fibrillation in normal and failing canine hearts

Because congestive heart failure (CHF) promotes ventricular fibrillation (VF), we compared VF in seven dogs with CHF induced by combined myocardial infarction and rapid ventricular pacing to VF in six normal dogs. A noncontact, multielectrode array balloon catheter provided full-surface real-time left ventricular (LV) endocardial electrograms and a dynamic color-coded display of endocardial activation projected onto a three-dimensional model of the LV. Fast Fourier transform (FFT) analysis of virtual electrograms showed no difference in peak or centroid frequency in CHF dogs compared with normals. The average number of simultaneous noncontiguous wavefronts present during VF was higher in normals (2.4 +/- 1.0 at 10 s of VF) than in CHF dogs (1.3 +/- 1.0, P < 0.005) and decreased in both over time. The wavefront "turnover" rate, estimated using FFT of the noncontiguous wavefront data, did not differ between normals and CHF and did not change over 5 min of VF. Thus the fundamental frequency characteristics of VF are unaltered by CHF, but dilated abnormal ventricles sustain fewer active wavefronts than do normal ventricles.     

Model for a helical bundle channel based on the high-resolution crystal structure of trichotoxin_A50E

Trichotoxin_A50E is an 18-residue peptaibol antibiotic which forms multimeric transmembrane channels through self-association. The crystal structure of trichotoxin has been determined at a resolution of 0.9 A. The trichotoxin sequence contains nine helix-promoting Aib residues, which contribute to the formation of an entirely helical structure that has a central bend of 8-10 degrees located between residues 10-13. Trichotoxin is the first solved structure of the peptaibol family that is all alpha-helix as opposed to containing part or all 3(10)-helix. Gln residues in positions 6 and 17 produce a polar face, and are proposed to form the channel lumen. An octameric model channel has been constructed from the crystal structure. It has a central pore of approximately 4-5 A radius, a size sufficient to enable transport of ions, with a constricted region at one end, formed by a ring of Gln6 residues. Electrostatic calculations are consistent with it being a cationic channel.     

Characterization of self-transmissible plasmids determining lactose fermentation and multiple antibiotic resistance in clinical strains of Klebsiella pneumoniae

The lactose fermentation (Lac+) and antibiotic resistance (R+) phenotypes were conjugally transferred from Klebsiella pneumoniae strains (K166, K182, K186, K218, and K220) to Salmonella typhi, S. typhimurium, Shigella flexneri, and Vibrio cholerae. The genes for lactose fermentation and antibiotic resistance were located on the plasmids. Further analysis of plasmid DNA from these isolates indicated the presence of multiple plasmids (Mr ranged less than 2.7 to 70 X 10(6)). The Lac+R+ plasmids p166 and p182 were members of the FII incompatibility group. The fertility inhibition property of plasmids, p182, p218, and p220 was fi+ type. Furthermore, phage typing experiments showed that plasmids p166 and p218 (Lac+R+) conferred the ability to inhibit the multiplication of bacteriophages 12 and 13 in S. typhimurium. However, the plasmids p182, p186, and p220 (Lac+R+) could inhibit the visible lysis of all the 30 phages in S. typhimurium. This study describes the characterization of Lac+R+ plasmids and the medical significance of an intergeneric transfer of lactose fermentation to non-lactose-fermenting pathogens.     

Evaluation of three serological tests for detection of anti-candidal antibodies in diagnosis of invasive candidiasis

Immunoblot detection of antibody against 47 KD cytoplasmic antigen ofCandida albicans was evaluated in diagnosis of invasive candidiasis and compared to whole cell agglutination and gel diffusion tests for detection of anticandidal antibody in 64 patients. The patients included 17 with culture proved candidemia, 34 with significant candiduria (more than 10,000 colony forming units per ml of urine) and 13 with nonsignificant candiduria. Antibody against 47 KD antigen was found to be the best indicator for diagnosis of invasive candidiasis even in patients with malignancy. The sensitivity of this procedure was 82.4%, specificity 86.7%, positive predictive value 77.8%, negative predictive value 89.7% and efficacy 85.1%. The gel diffusion procedure lacked in sensitivity whereas whole cell agglutination lacked in specificity. Detection of antibody against 47 KD antigen proved to be a valuable adjunct in the diagnosis of invasive candidiasis.     

Monophyly of the order Rodentia inferred from mitochondrial DNA sequences of the genes for 12S rRNA, 16S rRNA, and tRNA-valine

A recent analysis of amino acid sequence data (Graur et al.) suggested that the mammalian order Rodentia is polyphyletic, in contrast to most morphological data, which support rodent monophyly. At issue is whether the hystricognath rodents, such as the guinea pig, represent an independent evolutionary lineage within mammals, separate from the sciurognath rodents. To resolve this problem, we sequenced a region (2,645 bp) of the mitochondrial genome of the guinea pig containing the complete 12S ribosomal RNA, 16S ribosomal RNA, and transfer RNA(VAL) genes for comparison with the available sciurognath and other mammalian sequences. Several methods of analysis and statistical tests of the data all show strong support for rodent monophyly (91%-98% bootstrap probability, or BP). Calibration with the mammalian fossil record suggests a Cretaceous date (107 mya) for the divergence of sciurognaths and hystricognaths. An older date (38 mya) for the controversial Mus- Rattus divergence also is supported by these data. Our neighbor-joining analyses of all available sequence data (25 genes) confirm that some individual genes support rodent polyphyly but that tandem analysis of all data does not. We propose that the conflicting results are due to several compounding factors. The unique biochemical properties of some hystricognath metabolic proteins, largely responsible for generating this controversy, may have a single explanation: a cascade effect resulting from inactivation of the zinc-binding abilities of insulin. After excluding six genes possibly affected by insulin inactivation, analyses of all available sequence data (7,117 nucleotide sites, 3,099 amino acid sites) resulted in strong support for rodent monophyly (94% BP for DNA sequences, 90% for protein sequences), which lends support to the insulin-cascade hypothesis.      

Higher-level snake phylogeny inferred from mitochondrial DNA sequences of 12S rRNA and 16S rRNA genes

Portions of two mitochondrial genes (12S and 16S ribosomal RNA) were sequenced to determine the phylogenetic relationships among the major clades of snakes. Thirty-six species, representing nearly all extant families, were examined and compared with sequences of a tuatara and three families of lizards. Snakes were found to constitute a monophyletic group (confidence probability [CP] = 96%), with the scolecophidians (blind snakes) as the most basal lineages (CP = 99%). This finding supports the hypothesis that snakes underwent a subterranean period early in their evolution. Caenophidians (advanced snakes), excluding Acrochordus, were found to be monophyletic (CP = 99%). Among the caenophidians, viperids were monophyletic (CP = 98%) and formed the sister group to the elapids plus colubrids (CP = 94%). Within the viperids, two monophyletic groups were identified: true vipers (CP = 98%) and pit vipers plus Azemiops (CP = 99%). The elapids plus Atractaspis formed a monophyletic clade (CP = 99%). Within the paraphyletic Colubridae, the largely Holarctic Colubrinae was found to be a monophyletic assemblage (CP = 98%), and the Xenodontinae was found to be polyphyletic (CP = 91%). Monophyly of the henophidians (primitive snakes) was neither supported nor rejected because of the weak resolution of relationships among those taxa, except for the clustering of Calabaria with a uropeltid, Rhinophis (CP = 94%).