A novel property of hexokinase inhibition by Favipiravir and proposed advantages over Molnupiravir and 2 Deoxy D glucose in treating COVID-19.

PURPOSE: In the wake of SARS-CoV-2's global spread, human activities from health to social life to education have been affected. Favipiravir and Molnupiravir exhibited novel hexokinase inhibition and we discuss advantages of this property in their COVID-19 inhibition potential. METHODS: This paper describes molecular docking data of human hexokinase II with Favipiravir, Cyan 20, Remdesivir, 2DG, and Molnupiravir along with hexokinase inhibition assays. RESULTS: Favipiravir, an antiviral drug previously cleared for treating the flu and ebola, has shown some promise in early trials to treat COVID-19. We observed potent human hexokinase inhibiting potential of Favipiravir (50%) as against 4% and merely 0.3% hexokinase inhibition with Molnupiravir and 2 Deoxy D glucose at 0.1 mM concentration supported by molecular docking studies. CONCLUSION: Favipiravir could continue to be part of the COVID-19 treatment regimen due to its resistance to host esterases, hexokinase inhibition potential and proven safety through human trials.

G6PD deficiency and severity of COVID19 pneumonia and acute respiratory distress syndrome: tip of the iceberg?

The severe pneumonia caused by the human coronavirus (hCoV)-SARS-CoV-2 has inflicted heavy casualties, especially among the elderly and those with co-morbid illnesses irrespective of their age. The high mortality in African-Americans and males, in general, raises the concern for a possible X-linked mediated process that could affect the viral pathogenesis and the immune system. We hypothesized that G6PD, the most common X-linked enzyme deficiency, associated with redox status, may have a role in severity of pneumonia. Retrospective chart review was performed in hospitalized patients with COVID19 pneumonia needing supplemental oxygen. A total of 17 patients were evaluated: six with G6PD deficiency (G6PDd) and 11 with normal levels. The two groups (normal and G6PDd) were comparable in terms of age, sex, co-morbidities, and laboratory parameters-LDH, IL-6, CRP, and ferritin, respectively. Thirteen patients needed ventilatory support ; 8 in the normal group and 5 in the G6PDd group (72% vs.83%). The main differences indicating increasing severity in normal vs. G6PDd groups included G6PD levels (12.2 vs. 5.6, P = 0.0002), PaO2/FiO2 ratio (159 vs. 108, P = 0.05), days on mechanical ventilation (10.25 vs. 21 days P = 0.04), hemoglobin level (10 vs. 8.1 P = 0.03), and hematocrit (32 vs. 26 P = 0.015). Only one patient with G6PDd died; 16 were discharged home. Our clinical series ascribes a possible biological role for G6PDd in SARS-CoV2 viral proliferation. It is imperative that further studies are performed to understand the interplay between the viral and host factors in G6PDd that may lead to disparity in outcomes. KEY POINTS: • COVID19 studies show higher mortality in men, due to severe pneumonia and ARDS, indicating possible X-linked mediated differences • G6PD, the most common X-linked enzymopathy, highly prevalent in African Americans and Italians, maintains redox homeostasis. • Preclinical studies using G6PD deficient (G6PDd) cells infected with human coronavirus (hCoV), show impaired cellular responses, viral proliferation and worsening oxidative damage. • Retrospective chart review in hospitalized patients with COVID19 pneumonia needing supplemental oxygen shows differences between the two groups (Normal and G6PDd) in hematological indices; the G6PDdgroup demonstrated prolonged PaO2/FiO2 ratio, and longer days on mechanical ventilation indicating the severity of the pneumonia.

Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan.

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other ß-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

Uremic Pericarditis and Cardiac Tamponade Resolving With Intensive Hemodialysis.

We present an interesting and complex case of cardiac tamponade due to uremic pericarditis (UP), resolving with intensive hemodialysis (HD). HD should be considered as first line management for patients with UP and pericardial effusion. Intensification of HD should be considered based on clinical presentation and severity of presentation.

Development and Validation of Stability-Indicating Impurity Profiling Method for Azelastine Hydrochloride and Fluticasone Propionate in Nasal Spray Product Using HPLC with a UV/PDA Detector.

BACKGROUND: Azelastine HCl (AZ) and fluticasone propionate (FL) nasal spray drug product is commonly used in the treatment of allergic rhinitis worldwide. To date, the impurity profiling of this product has not been reported. OBJECTIVE: The present study aimed to develop and validate a novel RP-HPLC stability-indicating analytical method for the estimation of impurities from AZ and FL nasal spray drug product. METHODS: A mixture of octane sulfonic acid sodium salt and trifluroacetic acid is used as a mobile phase A. Acetonitrile is used as a mobile phase B. Good separation was achieved on Baker bond phenyl hexyl, 250 × 4.6, 5 µm column at 1 mL/min flow rate in gradient elution mode. The chromatograms were monitored at 239 nm. RESULTS: The LOD and LOQ were found to be 0.006 and 0.019 µg/mL for AZ and 0.010 and 0.030 µg/mL for FL, respectively. The correlation coefficient for all the known impurities and principal analytes was 0.999 from LOQ level to 150% of standard concentration. The recovery for all the known impurities was found to be between 90 and 110%. In the stress study, 15% degradation was observed in basic conditions and 8.7% in acidic conditions. No significant degradation was observed in thermal and oxidative conditions. CONCLUSION: An impurity profiling method for AZ and FL combination nasal spray product was successfully developed, validated, and demonstrated to be accurate, precise, specific, robust, and stability-indicating. The method can be routinely used for impurity testing of commercial batches in QC laboratories in the pharmaceutical industry. HIGHLIGHTS: No impurity study has been reported for this combination product until now.

Acute inferior ST-elevation myocardial infarction mimicked by direct lightning strike: a case report.

Background: Direct lightning strikes are rare, and multiple organ systems can be involved. Prognosis is dependent on the severity of the injury. Severe myocardial injury associated with transient electrocardiogram changes, which have been previously described, is a hazardous complication. Case summary: A 35-year-old man with no known past medical history presented unresponsive following a direct lightning strike while sitting in a portable toilet. High-quality cardiopulmonary resuscitation was started in the field, with return of spontaneous circulation (ROSC) after 1 h. Following ROSC, he received volume resuscitation and was maintained on multiple vasopressors. Electrocardiogram showed significant ST-elevations in inferior leads with elevated troponin I, consistent with inferior ST-elevation myocardial infarction. Labs revealed lactic acidosis, hyperkalaemia, acute kidney, and liver injury. Due to concern for plaque rupture, coronary angiography was performed and revealed no obstructive coronary artery disease. Vasopressor support and volume resuscitation were continued for extensive burns covering greater than 30% body surface area. The patient became progressively hypotensive, eventually precipitating pulseless electrical activity arrest. Emergent labs were notable for severe acidaemia. Despite aggressive interventions, he expired due to severe multi-organ failure. Discussion: Direct lightning injuries are rare with serious potential complications. Myocardial damage, either from direct electrical insult or from induced coronary vasospasm, can lead to multi-organ system failure.

Staphylococcus bacteriophage tails with bactericidal properties: new findings.

The development of lytic bacteriophages as therapeutic products is an attractive alternative to antibiotics. In this study, we evaluated the potential of phage tails for lysing Gram-positive bacteria. Phage P954, a well-characterized temperate staphylococcal phage, was found to adsorb to a large number of Staphylococcus aureus clinical isolates, although it lyses only 24% of the tested isolates. However, P954 phage tails generated by interruption of phage assembly were bactericidal against all the phage-resistant isolates. Phage tail preparations were trypsin sensitive with an apparent molecular weight of over 300 kDa. PCR analysis of the P954 phage-resistant isolates indicated the integration of P954-like prophages into the host genomes. Our study demonstrates for the first time that P954 bacteriophage tails have a much broader host range than the intact phage because phage tails are not affected by superinfection immunity or vulnerable to host restriction endonucleases.

Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection.

BACKGROUND: Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy. RESULTS: We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection. CONCLUSIONS: A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage effectively rescued mice from fatal S. aureus infection. To our knowledge this is the first report of a lysis-deficient staphylococcal phage.

A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME) from Phage K and its development into a potent antistaphylococcal protein.

BACKGROUND: Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. However, the rapid emergence of antibiotic resistance limits the choice of therapeutic options for treating infections caused by this organism. Muralytic enzymes from bacteriophages have recently gained attention for their potential as antibacterial agents against antibiotic-resistant gram-positive organisms. Phage K is a polyvalent virulent phage of the Myoviridae family that is active against many Staphylococcus species. RESULTS: We identified a phage K gene, designated orf56, as encoding the phage tail-associated muralytic enzyme (TAME). The gene product (ORF56) contains a C-terminal domain corresponding to cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), which demonstrated muralytic activity on a staphylococcal cell wall substrate and was lethal to S. aureus cells. We constructed N-terminal truncated forms of ORF56 and arrived at a 16-kDa protein (Lys16) that retained antistaphylococcal activity. We then generated a chimeric gene construct encoding Lys16 and a staphylococcal cell wall-binding SH3b domain. This chimeric protein (P128) showed potent antistaphylococcal activity on global clinical isolates of S. aureus including methicillin-resistant strains. In addition, P128 was effective in decolonizing rat nares of S. aureus USA300 in an experimental model. CONCLUSIONS: We identified a phage K gene that encodes a protein associated with the phage tail structure. The muralytic activity of the phage K TAME was localized to the C-terminal CHAP domain. This potent antistaphylococcal TAME was combined with an efficient Staphylococcus-specific cell-wall targeting domain SH3b, resulting in the chimeric protein P128. This protein shows bactericidal activity against globally prevalent antibiotic resistant clinical isolates of S. aureus and against the genus Staphylococcus in general. In vivo, P128 was efficacious against methicillin-resistant S. aureus in a rat nasal colonization model.

Cloning, soluble expression and purification of high yield recombinant hGMCSF in Escherichia coli.

Expression of human granulocyte macrophage colony stimulating factor (hGMCSF), a cytokine of therapeutic importance, as a thioredoxin (TRX) fusion has been investigated in Escherichia coli BL21 (DE3) codon plus cells. The expression of this protein was low when cloned under the T7 promoter without any fusion tags. High yield of GMCSF was achieved (∼88 mg/L of fermentation broth) in the shake flask when the gene was fused to the E. coli TRX gene. The protein was purified using a single step Ni(2+)-NTA affinity chromatography and the column bound fusion tag was removed by on-column cleavage with enterokinase. The recombinant hGMCSF was expressed as a soluble and biologically active protein in E. coli, and upon purification, the final yield was ∼44 mg/L in shake flask with a specific activity of 2.3 × 10(8) U/mg. The results of Western blot and RP-HPLC analyses, along with biological activity using the TF-1 cell line, established the identity of the purified hGMCSF. In this paper, we report the highest yield of hGMCSF expressed in E. coli. The bioreactor study shows that the yield of hGMCSF could be easily scalable with a yield of ∼400 mg/L, opening up new opportunities for large scale production hGMCSF in E. coli.

Case report of Barth syndrome: a forgotten cause of cardiomyopathy.

BACKGROUND: Barth syndrome (BTHS) is a rare X-linked recessive disorder characterized by clinical features including cardiomyopathy, skeletal myopathy, neutropenia, growth delay, and exercise intolerance. It is often considered to be a paediatric disease, owing to most cases being diagnosed during childhood and mortality being the highest during the first few years of life. CASE SUMMARY: We report a case of dilated cardiomyopathy due to BTHS in a 27-year-old adult male patient, who initially presented with lightheadedness, dyspnoea, orthopnoea, and bilateral lower extremity oedema. Key findings from investigations included leukopenia, prolonged QTc interval, reduced left ventricular ejection fraction (LVEF), global enlargement of all heart chambers, patent coronary arteries, and mild pulmonary hypertension. The patient was diuresed to euvolemia and discharged with a LifeVest. Guideline-directed medical therapy was initiated and uptitrated as an outpatient. A repeat echocardiogram 2 years after initial presentation showed marked improvement in LVEF. DISCUSSION: It is possible that there are adult patients with idiopathic cardiomyopathy, which may be attributable to BTHS. In the absence of an obvious underlying cause, with the appropriate historical information, clinical exam, laboratory investigations, and imaging findings, BTHS should be considered as a likely cause of non-ischaemic cardiomyopathy.

Novel self-cleavage activity of Staphylokinase fusion proteins: An interesting finding and its possible applications.

Staphylokinase (SAK) is reported to have a serine protease domain with no proteolytic activity unlike other plasminogen activators like tissue plasminogen activator (t-PA) and urokinase. A unique protease property of Staphylokinase was observed when SAK was expressed as a fusion protein in inducible Escherichia coli expression vectors. This finding was further investigated by cloning and expressing different SAK fusions, both native and N-terminal deletions, with fusion tags like glutathione S-transferase (GST) and signal sequence of SAK in bacterial system. While all the N-terminal SAK fusions were found to self-cleave in crude and purified preparations, the C-terminal SAK fusion was stable. The cleavage property of Staphylokinase fusion proteins, inhibited by reduced glutathione and PMSF, was independent of its thrombolytic activity and also independent on the type of host employed for its expression. The serine protease domain of the SAK gene possibly lies between 20th to 77th amino acid and serine 41 of this region appears critical for such a cleavage property.

Strategies to maximize expression of rightly processed human interferon alpha2b in Pichia pastoris.

The human interferon alpha 2b (IFN alpha2b) belongs to the interferon family of cytokines that exerts many biological functions like inhibition of virus multiplication, repression of tumour growth and other immunological functions. Herein, a synthetic gene coding for human IFN alpha2b was cloned and integrated into a methylotropic yeast-Pichiapastoris. The recombinant human IFN alpha2b protein (approximately 19kDa) could be successfully expressed in Pichiapastoris to a level of nearly 300mg/L with nearly 93% recovery on purification using a single anion exchange chromatography step. A novel media component dimethyl sulphoxide (DMSO) was found to aid in expression of rightly processed IFN alpha2b form with dramatic reduction in the expression of a 20kDa IFN isoform contaminant frequently observed by other workers. The identity of the 20kDa isoform was confirmed by N terminal sequencing which showed extra eleven amino acids at the N terminal portion of the IFN molecule obtained due to incorrect processing by the host KEX2 protease. The purified IFN alpha2b (19kDa) preparation was confirmed by N terminal sequencing, and characterized by MALDI-TOF and Agilent 2100 Bioanalyzer. The bioassay of the recombinant protein gave a specific activity of >2x10(8)IU/mg.

A novel prokaryotic vector for identification and selection of recombinants: direct use of the vector for expression studies in E. coli.

BACKGROUND: The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc. RESULTS: We describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP) as the reporter gene and is constructed in such a way that the E. coli cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts. CONCLUSIONS: This is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein.

Enhanced fluorescent properties of an OmpT site deleted mutant of green fluorescent protein.

BACKGROUND: The green fluorescent protein has revolutionized many areas of cell biology and biotechnology since it is widely used in determining gene expression and for localization of protein expression. Expression of recombinant GFP in E. coli K12 host from pBAD24M-GFP construct upon arabinose induction was significantly lower than that seen in E. coli B cells with higher expression at 30 degrees C as compared to 37 degrees C in E. coli K12 hosts. Since OmpT levels are higher at 37 degrees C than at 30 degrees C, it prompted us to modify the OmpT proteolytic sites of GFP and examine such an effect on GFP expression and fluorescence. Upon modification of one of the two putative OmpT cleavage sites of GFP, we observed several folds enhanced fluorescence of GFP as compared to unmodified GFPuv (Wild Type-WT). The western blot studies of the WT and the SDM II GFP mutant using anti-GFP antibody showed prominent degradation of GFP with negligible degradation in case of SDM II GFP mutant while no such degradation of GFP was seen for both the clones when expressed in BL21 cells. The SDM II GFP mutant also showed enhanced GFP fluorescence in other E. coli K12 OmpT hosts like E. coli JM109 and LE 392 in comparison to WT GFPuv. Inclusion of an OmpT inhibitor, like zinc with WT GFP lysate expressed from an E. coli K12 host was found to reduce degradation of GFP fluorescence by two fold. RESULTS: We describe the construction of two GFP variants with modified putative OmpT proteolytic sites by site directed mutagenesis (SDM). Such modified genes upon arabinose induction exhibited varied degrees of GFP fluorescence. While the mutation of K79G/R80A (SDM I) resulted in dramatic loss of fluorescence activity, the modification of K214A/R215A (SDM II) resulted in four fold enhanced fluorescence of GFP. CONCLUSIONS: This is the first report on effect of OmpT protease site modification on GFP fluorescence. The wild type and the GFP variants showed similar growth profile in bioreactor studies with similar amounts of recombinant GFP expressed in the soluble fraction of the cell. Our observations on higher levels of fluorescence of SDM II GFP mutant over native GFPuv in an OmpT+ host like DH5alpha, JM109 and LE392 at 37 degrees C reiterates the role played by host OmpT in determining differences in fluorescent property of the expressed GFP. Both the WT GFP and the SDM II GFP plasmids in E. coli BL21 cells showed similar expression levels and similar GFP fluorescent activity at 37 degrees C. This result substantiates our hypothesis that OmpT protease could be a possible factor responsible for reducing the expression of GFP at 37 degrees C for WT GFP clone in K12 hosts like DH5alpha, JM109, LE 392 since the levels of GFP expression of SDM II clone in such cells at 37 degrees C is higher than that seen with WT GFP clone at the same temperature.

Benefits of Soleris® over the Conventional Method for Enumeration of Microbial Load in Salacia Herbal Extract.

Stems and roots of Salacia genus plants have been used as a specific remedy for early-stage diabetes, and one of the four sulphonium sulphates, salacinol is the compound responsible for the anti-diabetic activity. Salacia is prone to microbial contamination and insect infestation; hence, methods to estimate the microbial load in such plants will enhance its nutritional value. This paper highlights the novel use of Soleris® to quantify microbes of all types, namely bacteria, yeasts, molds, and coliforms in herbal extracts. The microbial analysis results obtained with Soleris® test vial have been compared with the conventional method, and the results indicate that Soleris® is equally efficient as the conventional method and in fact displays several advantages over the traditional method. The Soleris® method is a real time monitoring system that is highly sensitive, user-friendly, and environmentally friendly since it generates very little biomedical waste and saves a large amount of time. The data presented here demonstrate that for highly contaminated samples, results are available within 24 h. For yeasts and molds, the Soleris® method produces results in 48 h, thus offering considerable time savings compared to other commonly used methods.

High yielding recombinant Staphylokinase in bacterial expression system--cloning, expression, purification and activity studies.

Staphylokinase (SAK) is emerging as an important thrombolytic agent. In this report, we describe the cloning, expression, purification and activity studies of the SAK gene of Staphylococcus aureus from a custom synthesised SAK gene. The SAK gene of 411 bp yielded a protein of approximately 15 kDa when expressed under pET21a vector using IPTG as an inducer in BL21 (DE3) pLysE codon Plus cells. The recombinant SAK (rSAK) was soluble in nature and constituted nearly 35% of the total cellular protein as estimated by densitometry scanning. Fermentation studies were carried out to optimize various parameters for maximizing the yield of rSAK and with the optimized medium, the yield of rSAK was nearly 2.8 g/L of fermentation broth, which is highest yield of rSAK expressed in any bacterial system till date. Two simple purification steps of ion-exchange chromatography yielded homogenous rSAK with almost 36% recovery. The purified SAK protein was characterized by MALDI-TOF and by plasminogen activation studies. The rSAK was found to be active by the chromogenic substrate assay method.

Efficient expression and secretion of recombinant human growth hormone in the methylotrophic yeast Pichia pastoris: potential applications for other proteins.

A simple high yielding process for the production of rhGH (recombinant human growth hormone) in the Pichia pastoris system is described. The approach adopted the addition of surfactants during fermentation along with methanol induction. A Pichia integrant harbouring multiple-copy, non-codon-optimized hGH showed poor expression in complex and defined media. Inclusion of the surfactants Tween 20 or Tween 80 during induction enhanced the expression levels significantly in shake flask studies. The combination of 2 litres of basal salt medium and Tween 20 in a bioreactor culminated in 3 x 10(4)-fold elevated expression of the protein (approximately 500 mg/l) as estimated by ELISA. SDS/PAGE and Western-blot analyses revealed that the Pichia-derived rhGH migrated as a single band with a molecular mass of approximately 22 kDa and had the same immunoreactivity as native commercial rhGH. Analysis of Pichia-derived purified rhGH and commercial rhGH on an Agilent 2100 Bioanalyzer revealed overlapping peaks displaying authentic N-terminal processing of Pichia-derived rhGH, which was further confirmed by N-terminal sequencing. In addition, matrix-assisted laser-desorption ionization-time of flight analysis of the protein confirmed its authenticity. These results indicate that the P. pastoris expression system can be effective in the production of rhGH at commercially relevant levels.

Construction of a novel zero background prokaryotic expression vector: potential advantages.

A novel DNA sequence, derived from the antisense strand of the DNA gyrase inhibitor protein, CcdB, was toxic to E. coli. This protein (approximately 6 kDa) decreased the growth rate of E. coli K12 by three orders of magnitude upon induction. The expressed toxic protein in E. coli K12 was soluble while it was insoluble in induced E. coli BL21. A high efficiency prokaryotic cloning/expression vector was constructed using this toxic gene sequence and gave zero background with approximately 100% cloning efficiency requiring no dephosphorylation. The toxic gene product also affected the survival of a ccdB resistant cell line, thus indicating a different mechanism of toxicity, other than DNA gyrase inhibition, as compared to the ccdB toxicity.

High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris.

Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (approximately 1 g/l) of extracellular glycosylated rSAK (approximately 18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (approximately 15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.