scholarly journals High-yield Production of Recombinant Platelet Factor 4 Protein by Harnessing and Honing the Gram-negative Bacterial Secretory Apparatus

2019 ◽  
Author(s):  
Saeed Ataei ◽  
Mohammad Naser Taheri ◽  
Fatemeh Taheri ◽  
Farahnaz Zare ◽  
Niloofar Amirian ◽  
...  
Keyword(s):  
Light Scattering ◽  
Secondary Structure ◽  
Protein Secretion ◽  
Type I ◽  
Type Ii ◽  
Platelet Factor ◽  
Gram Negative ◽  
Extracellular Milieu ◽  
Secretory System ◽  
Triton X

AbstractBackgroundPlatelet factor 4 is a cytokine released into the bloodstream by activated platelets and plays a pivotal role in heparin-induced thrombocytopenia etiology and diagnosis. Therefore, a sustainable source of recombinant PF4 with structural and functional similarity to its native form is urgently needed to be used in diagnostic procedures.To this end, a three-in-one primary construct was designed and custom synthesized based on the pET26b backbone from which three secondary constructs could be derived each capable of employing either type I, type II secretory or cytoplasmic pathways. Protein expression and secretion were performed in Escherichia coli BL-21 (DE3) and were confirmed by SDS-PAGE and Western blotting. To further enhance protein secretion, the effect of several controllable factors including IPTG, Triton X-100, Sucrose, and Glycine were individually investigated at first. In the next step, according to fractional factorial approach, the synergistic effect of IPTG, Triton X-100, and Glycine on secretion was further investigated. To ascertain the structure and function of the secreted recombinant proteins, Dynamic light scattering was utilized and confirmed rPF4 tetramerization and heparin-mediated ultra-large complex formation. Moreover, Raman spectroscopy was exploited to determine the rPF4 secondary structure.ResultsType II secretory pathway was proven to be superior over type I in case of rPF4 secretion into the extracellular milieu. Protein secretion mediated by Type II was enhanced to approximately more than 700 μg/ml. Large quantities of native rPF4 up to 20 mg was purified upon a minor scale up to 40 ml of culture medium. Dynamic light scattering unveiled native rPF4 quaternary structure revealing the formation of tetramers having an average size of 10 nm and formation of larger complexes of approximately 100-1200 nm in size following heparin supplementation, implying proper protein folding, tetramerization, and antigenicity. Analysis of the Zeta potential on approximately 600 μg/ml of rPF4 revealed a 98 mV positive charge which further confirms protein folding. Moreover, rPF4 secondary structure was determined to be 43.5% Random coil, 32.5% β-sheet, 18.6 % α-helix and 4.9 % Turn, which is in perfect agreement with the native structure.Conclusionour results indicate that the gram-negative type II bacterial secretory system holds a great promise to be employed as a reliable protein production strategy with favorable industrial application. However, further efforts are required to realize the full potential of secretory pathways regarding their application to proteins with distinct characteristics.Abstract FigureGraphical Abstract.rPF4 secretion mediated by type 2 secretory system. The pelB signal sequence directs protein export into the extracellular milieu through the SecYEG translocon complex in a process assisted by SecB chaperone. A) Indicates protein secretion before supplementation with additives and B) indicates secretion in the presence of additives.

scholarly journals Effects of detergent on the sulphation of chondroitin by cell-free preparations from chick-embryo epiphyseal cartilage

1992 ◽  
Vol 285 (2) ◽  
pp. 577-583 ◽  
Author(s):  
G Sugumaran ◽  
J E Silbert
Keyword(s):  
Chick Embryo ◽  
Supernatant Fraction ◽  
Random Pattern ◽  
Type I ◽  
Type Ii ◽  
Enzyme Preparations ◽  
Enzyme Substrate ◽  
Ionic Detergent ◽  
The Individual ◽  
Triton X

The effects of the non-ionic detergent Triton X-100 on 6-sulphation of two species of endogenous nascent proteochondroitin by a chick-embryo cartilage microsomal system was examined. Sulphation of the larger (Type I) species with adenosine 3′-phosphate 5′-phosphosulphate was slightly diminished when Triton X-100 was present, whereas sulphation of the smaller (Type II) species was slightly enhanced. An ordered rather than random pattern of sulphation was obtained for the smaller proteoglycan, but with a considerably lower degree of sulphation than that of the larger proteochondroitin. These differences were consistent with other differences between these two species as described previously. Sulphation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system with Triton X-100 present produced ordered rather than random sulphation patterns. When a 100,000 g supernatant fraction was utilized for sulphation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and ordered sulphation was still obtained. When hexasaccharide was used, sulphation of multiple N-acetylgalactosamine residues of the individual hexasaccharides resulted. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulphation of multiple N-acetylgalactosamine residues on the individual hexasaccharide molecules was even greater, so that tri-sulphated products were found. This suggests that ordered rather than random sulphation of chondroitin with these enzyme preparations is due to enzyme-substrate interaction rather than to membrane organization.

scholarly journals Type I and Type II mechanisms of antimicrobial photodynamic therapy: An in vitro study on gram-negative and gram-positive bacteria

2012 ◽  
Vol 44 (6) ◽  
pp. 490-499 ◽  
Author(s):  
Liyi Huang ◽  
Yi Xuan ◽  
Yuichiro Koide ◽  
Timur Zhiyentayev ◽  
Masamitsu Tanaka ◽  
...  
Keyword(s):  
Photodynamic Therapy ◽  
In Vitro Study ◽  
Vitro Study ◽  
Type I ◽  
Antimicrobial Photodynamic Therapy ◽  
Type Ii ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

scholarly journals Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and β-sheet topology with the A chain of ricin, a type-II RIP

2008 ◽  
Vol 9 (1) ◽  
pp. 138-144 ◽  
Author(s):  
Yun-Xing Wang ◽  
Jaison Jacob ◽  
Dennis A. Torchia ◽  
Paul T. Wemgfteld ◽  
Ira Palmer ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Single Strand ◽  
Type I ◽  
Type Ii ◽  
A Chain ◽  
Β Sheet ◽  
Anti Hiv ◽  
Ricin A

scholarly journals Photosynthetic Systems Suggest an Evolutionary Pathway to Diderms

2020 ◽  
Author(s):  
Scott O. Rogers
Keyword(s):  
Reaction Centers ◽  
Type I ◽  
Type Ii ◽  
Evolutionary Pathway ◽  
Gram Negative ◽  
Evolutionary Changes ◽  
Alpha Beta ◽  
Taxonomic Groups ◽  
Peptidoglycan Layer ◽  
Molecular Characters

AbstractBacteria are divided primarily into monoderms (with one cell membrane, and usually Gram-positive, due to a thick peptidoglycan layer) and diderms (with two cell membranes, and mostly Gram-negative, due to a thin peptidoglycan layer sandwiched between the two membranes). Photosynthetic species are spread among the taxonomic groups, some having type I reaction centers (RCI in monoderm phylum Firmicutes; and diderm phyla Acidobacteria and Chlorobi), others with type II reaction centers (RCII in monoderm phylum Chloroflexi; and diderm taxa Gemmatimonadetes, and alpha-, beta-, and gamma-Proteobacteria), and some containing both (RCI and RCII, only in diderm phylum Cyanobacteria). In most bacterial phylograms, photosystem types and diderm taxa are polyphyletic. A more parsimonious arrangement, which is supported by photosystem evolution, as well as additional sets of molecular characters, suggests that endosymbiotic events resulted in the formation of the diderms. In the model presented, monoderms readily form a monophyletic group, while diderms are produced by at least two endosymbiotic events, followed by additional evolutionary changes.

scholarly journals Characterization of the protein kinase activities of human platelet supernatant and particulate fractions

1984 ◽  
Vol 218 (2) ◽  
pp. 285-294 ◽  
Author(s):  
S E Salama ◽  
R J Haslam
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Soluble Fraction ◽  
Deae Cellulose ◽  
Type I ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Regulatory Subunits ◽  
Dependent Protein ◽  
Triton X

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.

scholarly journals Formation of two species of nascent proteochondroitin in separate loci of a microsomal preparation from chick-embryo epiphyseal cartilage

1991 ◽  
Vol 277 (3) ◽  
pp. 787-793 ◽  
Author(s):  
G Sugumaran ◽  
J E Silbert
Keyword(s):  
Chick Embryo ◽  
Type I ◽  
Type Ii ◽  
Microsomal Preparation ◽  
Intact Membrane ◽  
Ionic Detergent ◽  
Before And After ◽  
Microsomal Membranes ◽  
Triton X ◽  
Relationship Of

The potential relationship of an intact membrane organization to the synthesis of chondroitin was examined before and after modification of a chick-embryo cartilage microsomal system with the non-ionic detergent Triton X-100. Incubations with labelled UDP-GlcA and UDP-GalNAc indicated that Triton X-100 had little effect on the amount of chondroitin synthesized to form one species of large proteochondroitin (Type I). However, Triton X-100 had a marked stimulatory effect on the formation of another smaller species of proteochondroitin (Type II). Presence of this detergent during chondroitin polymerization also resulted in chains that were slightly smaller. Neither of the two proteochondroitin species were collagenase-sensitive, nor did they contain dermatan-like regions. Thus in these respects they were unlike the small proteochondroitins (PG-Lb or PG-Lt) that have been found in chick-embryo cartilage. They also differed greatly in size from these small proteoglycans as well as from the large aggregatable proteochondroitin (PG-H) from the same source. Synthesis of the larger (Type I) proteochondroitin species was not affected by prior treatment of the microsomes with chondroitin ABC lyase at concentrations sufficient for elimination of synthesis of most of the smaller (Type II) proteochondroitin species. Use of chondroitin ABC lyase subsequent to synthesis of the chondroitin also resulted in preferential degradation of the smaller species. Thus there were differences in formation and limitation in access of the chondroitin ABC lyase to the two species, consistent with other differences described previously. These results indicate that there are separate loci within the microsomal membranes for synthesis of the two species.

scholarly journals Legionella pneumophila Type II Protein Secretion Promotes Virulence in the A/J Mouse Model of Legionnaires' Disease Pneumonia

2004 ◽  
Vol 72 (1) ◽  
pp. 310-321 ◽  
Author(s):  
Ombeline Rossier ◽  
Shawn R. Starkenburg ◽  
Nicholas P. Cianciotto
Keyword(s):  
Protein Secretion ◽  
Legionella Pneumophila ◽  
Complete Analysis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Kinetic Assay ◽  
Genome Database ◽  
Gram Negative ◽  
Legionnaires Disease

ABSTRACT Legionella pneumophila, the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase. By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans-complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L. pneumophila infection of both amoebae and human macrophages. Based upon a complete analysis of lspDE, lspF, and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies. Examination of the developing L. pneumophila genome database indicated that the organism has two other loci (lspC and lspLM) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria. In contrast to lsp mutants, L. pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication. L. pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes. Upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays. The lspF mutant was also defective in an in vivo kinetic assay. Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo. Thus, the L. pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.

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