scholarly journals Transcriptomics-Based Identification of Novel Factors Enhancing Heterologous Protein Secretion in Yeasts

2007 ◽  
Vol 73 (20) ◽  
pp. 6499-6507 ◽  
Author(s):  
Brigitte Gasser ◽  
Michael Sauer ◽  
Michael Maurer ◽  
Gerhard Stadlmayr ◽  
Diethard Mattanovich
Keyword(s):  
Protein Secretion ◽  
Rational Solution ◽  
Antibody Fragment ◽  
Efficient Production ◽  
Human Antibody ◽  
Scale Production ◽  
Heterologous Proteins ◽  
Fab Fragment ◽  
Folding Rate ◽  
Atpase Subunit

ABSTRACT Efficient production of heterologous proteins with yeasts and other eukaryotic hosts is often hampered by inefficient secretion of the product. Limitation of protein secretion has been attributed to a low folding rate, and a rational solution is the overexpression of proteins supporting folding, like protein disulfide isomerase (Pdi), or the unfolded protein response transcription factor Hac1. Assuming that other protein factors which are not directly involved in protein folding may also support secretion of heterologous proteins, we set out to analyze the differential transcriptome of a Pichia pastoris strain overexpressing human trypsinogen versus that of a nonexpressing strain. Five hundred twenty-four genes were identified to be significantly regulated. Excluding those genes with totally divergent functions (like, e.g., core metabolism), we reduced this number to 13 genes which were upregulated in the expression strain having potential function in the secretion machinery and in stress regulation. The respective Saccharomyces cerevisiae homologs of these genes, including the previously characterized secretion helpers PDI1, ERO1, SSO2, KAR2/BiP, and HAC1 as positive controls, were cloned and overexpressed in a P. pastoris strain expressing a human antibody Fab fragment. All genes except one showed a positive effect on Fab fragment secretion, as did the controls. Six out of these novel secretion helper factors, more precisely Bfr2 and Bmh2 (involved in protein transport), the chaperones Ssa4 and Sse1, the vacuolar ATPase subunit Cup5, and Kin2 (a protein kinase connected to exocytosis), proved their benefits for practical application in laboratory-scale production processes by increasing both specific production rates and the volumetric productivity of an antibody fragment up to 2.5-fold in fed-batch fermentations of P. pastoris.

A High-Affinity Human Antibody That Targets Tumoral Blood Vessels

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 192-198 ◽  
Author(s):  
Lorenzo Tarli ◽  
Enrica Balza ◽  
Francesca Viti ◽  
Laura Borsi ◽  
Patrizia Castellani ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Small Cell Lung Carcinoma ◽  
Antibody Fragment ◽  
Human Colon ◽  
Experimental Models ◽  
Human Antibody ◽  
Cell Lung Carcinoma ◽  
High Affinity ◽  
Biodistribution Studies

Angiogenesis is a characteristic feature of many aggressive tumors and of other relevant disorders. Molecules capable of specifically binding to new-forming blood vessels, but not to mature vessels, could be used as selective vehicles and would, therefore, open diagnostic and therapeutic opportunities. We have studied the distribution of the ED-B oncofetal domain of fibronectin, a marker of angiogenesis, in four different tumor animal models: the F9 murine teratocarcinoma, SKMEL-28 human melanoma, N592 human small cell lung carcinoma, and C51 human colon carcinoma. In all of these experimental models we observed accumulation of the fibronectin isoform containing the ED-B domain around neovascular structures when the tumors were in the exponentially growing phase, but not in the slow-growing phase. Then we performed biodistribution studies in mice bearing a subcutaneously implanted F9 murine teratocarcinoma, using a high-affinity human antibody fragment (L19) directed against the ED-B domain of fibronectin. Radiolabeled L19, but not an irrelevant anti-lysozyme antibody fragment (D1.3), efficiently localizes in the tumoral vessels. The maximal dose of L19 accumulated in the tumor was observed 3 hours after injection (8.2% injected dose per gram). By virtue of the rapid clearance of the antibody fragment from the circulation, tumor-to-blood ratios of 1.9, 3.7, and 11.8 were obtained at 3, 5, and 24 hours, respectively. The tumor-targeting performance of L19 was not dose-dependent in the 0.7 to 10 μg range of injected antibody. The integral of the radioactivity localized in tumoral vessels over 24 hours was greater than 70-fold higher than the integral of the radioactivity in blood over the same time period, normalized per gram of tissue or fluid. These findings quantitatively show that new-forming blood vessels can selectively be targeted in vivo using specific antibodies, and suggest that L19 may be of clinical utility for the immunoscintigraphic detection of angiogenesis in patients.

scholarly journals Dynamic consolidated bioprocessing for innovative lab-scale production of bacterial alkaline phosphatase from Bacillus paralicheniformis strain APSO

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Soad A. Abdelgalil ◽  
Nadia A. Soliman ◽  
Gaber A. Abo-Zaid ◽  
Yasser R. Abdel-Fattah
Keyword(s):  
Alkaline Phosphatase ◽  
Consolidated Bioprocessing ◽  
Black Liquor ◽  
Batch Cultivation ◽  
Efficient Production ◽  
Scale Production ◽  
Market Demand ◽  
Stirred Tank Bioreactor ◽  
Bacterial Alkaline Phosphatase ◽  
Bacillus Paralicheniformis

AbstractTo meet the present and forecasted market demand, bacterial alkaline phosphatase (ALP) production must be increased through innovative and efficient production strategies. Using sugarcane molasses and biogenic apatite as low-cost and easily available raw materials, this work demonstrates the scalability of ALP production from a newfound Bacillus paralicheniformis strain APSO isolated from a black liquor sample. Mathematical experimental designs including sequential Plackett–Burman followed by rotatable central composite designs were employed to select and optimize the concentrations of the statistically significant media components, which were determined to be molasses, (NH4)2NO3, and KCl. Batch cultivation in a 7-L stirred-tank bioreactor under uncontrolled pH conditions using the optimized medium resulted in a significant increase in both the volumetric and specific productivities of ALP; the alkaline phosphatase throughput 6650.9 U L−1, and µ = 0.0943 h−1; respectively, were obtained after 8 h that, ameliorated more than 20.96, 70.12 and 94 folds compared to basal media, PBD, and RCCD; respectively. However, neither the increased cell growth nor enhanced productivity of ALP was present under the pH-controlled batch cultivation. Overall, this work presents novel strategies for the statistical optimization and scaling up of bacterial ALP production using biogenic apatite.

Selective targeting and photocoagulation of ocular angiogenesis mediated by a phage-derived human antibody fragment

10.1038/13679 ◽  
1999 ◽  
Vol 17 (10) ◽  
pp. 984-988 ◽  
Author(s):  
Manfred Birchler ◽  
Francesca Viti ◽  
Luciano Zardi ◽  
Bernhard Spiess ◽  
Dario Neri
Keyword(s):  
Antibody Fragment ◽  
Human Antibody ◽  
Ocular Angiogenesis ◽  
Selective Targeting

scholarly journals Structure-Based Modification of an Anti-neuraminidase Human Antibody Restores Protection Efficacy against the Drifted Influenza Virus

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Haihai Jiang ◽  
Weiyu Peng ◽  
Jianxun Qi ◽  
Yan Chai ◽  
Hao Song ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Influenza Virus ◽  
Antibody Fragment ◽  
Salt Bridge ◽  
Cross Reactivity ◽  
Human Antibody ◽  
Influenza Strain ◽  
Group 2 ◽  
Fragment Antigen Binding ◽  
Group 1

ABSTRACT Here, we investigate a monoclonal antibody, Z2B3, isolated from an H7N9-infected patient, that exhibited cross-reactivity to both N9 (group 2) and a broad range of seasonal and avian N1 (group 1) proteins but lost activity to the N1 with the substitution K432E. This substitution exists in 99.25% of seasonal influenza strains after 2013. The NA-Z2B3 complex structures indicated that Z2B3 binds within the conserved active site of the neuraminidase (NA) protein. A salt bridge between D102 in Z2B3 and K432 in NA plays an important role in binding. Structure-based modification of Z2B3 with D102R in heavy chain reversed the salt bridge and restored the binding and inhibition of N1 with E432. Furthermore, Z2B3-D102R can protect mice from A/Serbia/NS-601/2014 H1N1 virus (NA contains E432) infection while the wild-type Z2B3 antibody shows no protection. This study demonstrates that a broadly reactive and protective antibody to NA can be in principle edited to restore binding and inhibition to recently drifted N1 NA and regain protection against the variant influenza strain. IMPORTANCE The immune system produces antibodies to protect the human body from harmful invaders. The monoclonal antibody (MAb) is one kind of effective antivirals. In this study, we isolated an antibody (Z2B3) from an H7N9 influenza virus-infected child. It shows cross-reactivity to both group 1 (N1) and group 2 (N9) neuraminidases (NAs) but is sensitive to N1 NA with a K432E substitution. Structural analysis of the NA-antibody fragment antigen-binding (Fab) complex provides a clue for antibody modification, and the modified antibody restored binding and inhibition to recently drifted N1 NA and regained protection against the variant influenza strain. This finding suggests that antibodies to NA may be a useful therapy and can be in principle edited to defeat drifted influenza virus.

scholarly journals Efficient Production of l-Ribose with a Recombinant Escherichia coli Biocatalyst

2008 ◽  
Vol 74 (10) ◽  
pp. 2967-2975 ◽  
Author(s):  
Ryan D. Woodyer ◽  
Nathan J. Wymer ◽  
F. Michael Racine ◽  
Shama N. Khan ◽  
Badal C. Saha
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Active Form ◽  
Apium Graveolens ◽  
Efficient Production ◽  
Scale Production ◽  
Gene Encoding ◽  
Large Scale Production ◽  
One Step ◽  
Rare Sugars

ABSTRACT A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 μM ZnCl2 and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter−1 day−1. This system represents a significantly improved method for the large-scale production of l-ribose.

Depletion of circulating α2-antiplasmin by intravenous plasmin or immunoneutralization reduces focal cerebral ischemic injury in the absence of arterial recanalization

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3086-3092 ◽  
Author(s):  
Nobus Nagai ◽  
Maria De Mol ◽  
Berthe Van Hoef ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Antibody Fragment ◽  
Ischemic Injury ◽  
Neutralizing Antibody ◽  
Human Beings ◽  
Fab Fragment ◽  
Single Bolus ◽  
Arterial Recanalization ◽  
Cerebral Ischemic Injury ◽  
Cerebral Ischemic ◽  
Single Bolus Injection

Abstract In the absence of arterial recanalization, thrombolytic agents induce a dose-related extension of focal cerebral ischemic injury (FII) in experimental animals. However, FII is smaller in mice lacking α2-antiplasmin (α2-AP), the physiologic inhibitor of plasmin, suggesting its depletion might reduce FII in the absence of reperfusion. Therefore, the effect of human plasmin (Pli), human miniplasmin (mPli), and an Fab fragment neutralizing murine α2-AP (Fab-4H9) on FII after middle cerebral artery (MCA) ligation was studied in mice and in hamsters. In BALB/c mice, the median FII after 24 hours was 28 μL (range, 20-34) (n = 10) with saline and 23 μL (range, 17-26) (n = 9) with a single bolus of 0.07 mg Pli, given after MCA ligation (P = .010), which reduced α2-AP to 44% and fibrinogen from 0.75 to 0.44 g/L. FII was 20 μL (range, 13-26) (n = 6, P = .025) with 0.2 mg mPli and was 24 μL (range, 20-27) (n = 6,P = .020) with 1.7 mg Fab-4H9. Neuronal atrophy and reduction of laminin immunoreactivity were comparably observed in the infarct area after saline and Pli. In hamsters, a single bolus injection of 1 mg Pli, after MCA ligation, depleted α2-AP and fibrinogen and reduced FII at 24 hours from 20 μL (range, 9.9-38) (n = 6) to 7.0 μL (range, 0.44-31) (n = 7,P = .032). Thus, reduction of circulating α2-AP, with a single bolus of plasmin or of a neutralizing antibody fragment, significantly reduced FII after MCA ligation in mouse and hamster models, suggesting that, provided these observations can be extrapolated to human beings, transient depletion of circulating α2-AP might reduce ischemic stroke in the absence of reperfusion.

scholarly journals Structure of the Fab Fragment of F105, a Broadly Reactive Anti-Human Immunodeficiency Virus (HIV) Antibody That Recognizes the CD4 Binding Site of HIV Type 1 gp120

2005 ◽  
Vol 79 (20) ◽  
pp. 13060-13069 ◽  
Author(s):  
Royce A. Wilkinson ◽  
Chayne Piscitelli ◽  
Martin Teintze ◽  
Lisa A. Cavacini ◽  
Marshall R. Posner ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Binding Site ◽  
Neutralizing Antibody ◽  
Binding Pocket ◽  
Human Antibody ◽  
Fab Fragment ◽  
Outer Domain ◽  
Immunodeficiency Virus ◽  
Cd4 Binding Site ◽  
The Difference

ABSTRACT We have determined the crystal structure of the Fab fragment from F105, a broadly reactive human antibody with limited potency that recognizes the CD4 binding site of gp120. The structure reveals an extended CDR H3 loop with a phenylalanine residue at the apex and shows a striking pattern of serine and tyrosine residues. Modeling the interaction between gp120 and F105 suggests that the phenylalanine may recognize the binding pocket of gp120 used by Phe43 of CD4 and that numerous tyrosine and serine residues form hydrogen bonds with the main chain atoms of gp120. A comparison of the F105 structure to that of immunoglobulin G1 b12, a much more potent and broadly neutralizing antibody with an overlapping epitope, suggests similarities that contribute to the broad recognition of human immunodeficiency virus by both antibodies. While the putative epitope for F105 shows significant overlap with that predicted for b12, it appears to differ from the b12 epitope in extending across the interface between the inner and outer domains of gp120. In contrast, the CDR loops of b12 appear to interact predominantly with the outer domain of gp120. The difference between the predicted epitopes for b12 and F105 suggests that the unique potency of b12 may arise from its ability to avoid the interface between the inner and outer domains of gp120.

Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin

2001 ◽  
Vol 28 (4) ◽  
pp. 534-539 ◽  
Author(s):  
Salvatore Demartis ◽  
Lorenzo Tarli ◽  
Laura Borsi ◽  
Luciano Zardi ◽  
Dario Neri
Keyword(s):  
Antibody Fragment ◽  
Human Antibody ◽  
Selective Targeting

scholarly journals Efficient production of antibody Fab fragment by transient gene expression in insect cells

2017 ◽  
Vol 124 (2) ◽  
pp. 221-226 ◽  
Author(s):  
Keita Mori ◽  
Hirotsugu Hamada ◽  
Takafumi Ogawa ◽  
Yuki Ohmuro-Matsuyama ◽  
Tomohisa Katsuda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Insect Cells ◽  
Transient Gene Expression ◽  
Efficient Production ◽  
Fab Fragment

A neutralizing recombinant human antibody Fab fragment against Puumala hantavirus

2000 ◽  
Vol 60 (4) ◽  
pp. 446-454 ◽  
Author(s):  
Cristina de Carvalho Nicacio ◽  
�ke Lundkvist ◽  
Katarina Brus Sj�lander ◽  
Alexander Plyusnin ◽  
Eeva-Marjatta Salonen ◽  
...  
Keyword(s):  
Human Antibody ◽  
Fab Fragment
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