scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2020 ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Membrane Vesicles ◽  
Lipid Vesicles ◽  
Protein Glycosylation ◽  
Glycoprotein Synthesis ◽  
Membrane Mimics ◽  
Native Cell ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membranebound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3×1012 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7×1012 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

scholarly journals Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli

1981 ◽  
Vol 200 (3) ◽  
pp. 611-627 ◽  
Author(s):  
K R Daruwalla ◽  
A T Paxton ◽  
P J Henderson
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Membrane Vesicles ◽  
Kinetic Studies ◽  
Transport Systems ◽  
Protonmotive Force ◽  
Alkaline Ph ◽  
Ph Change ◽  
Intact Cells ◽  
Membrane Bound

1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.

scholarly journals Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene

2002 ◽  
Vol 364 (3) ◽  
pp. 795-805 ◽  
Author(s):  
Adrian P. BROWN ◽  
Simon CARNABY ◽  
Clare BROUGH ◽  
Melissa BRAZIER ◽  
Antoni R. SLABAS
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Lysophosphatidic Acid ◽  
Leaf Tissue ◽  
Acid Synthesis ◽  
Protein Antigens ◽  
Maximal Level ◽  
E Coli ◽  
Developing Seeds ◽  
Membrane Bound

Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27pg/μg of membrane protein in leaf tissue and ≤ 12.5pg/μg of membrane protein in developing embryos. The amount of LAT2 reaches a peak at 305pg/μg of membrane protein 25daf and is not expressed 20daf or before. This is the first study to quantify these membrane-bound proteins in a plant tissue. The maximal level of LAT2 protein coincides with the maximal level of erucic acid synthesis in the seeds. Both full-length proteins were expressed in the Escherichia coli LPAAT mutant JC201, and membranes from these strains were used to investigate the substrate selectivity of these two enzymes, demonstrating that they are different. Finally, we report that LAT2 and a maize LPAAT enzyme (MAT1) can functionally replace the E. coli plsC gene after its deletion in the chromosome, whereas LAT1 and a coconut LPAAT (Coco1) cannot. This is probably due to differences in substrate utilization.

scholarly journals Immunization With Outer Membrane Vesicles Derived From Major Outer Membrane Protein-Deficient Salmonella Typhimurium Mutants for Cross Protection Against Salmonella Enteritidis and Avian Pathogenic Escherichia coli O78 Infection in Chickens

2020 ◽  
Vol 11 ◽  
Author(s):  
Yuxuan Chen ◽  
Kaiwen Jie ◽  
Biaoxian Li ◽  
Haiyan Yu ◽  
Huan Ruan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Broad Spectrum ◽  
Outer Membrane Protein ◽  
Salmonella Enteritidis ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Major Outer Membrane Protein ◽  
Cross Protection

Colibacillosis is an economically important infectious disease in poultry, caused by avian pathogenic Escherichia coli (APEC). Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne diseases in human circulated through poultry-derived products, including meat and chicken eggs. Vaccine control is the mainstream approach for combating these infections, but it is difficult to create a vaccine for the broad-spectrum protection of poultry due to multiple serotypes of these pathogens. Our previous studies have shown that outer membrane vesicles (OMVs) derived from S. enterica serovar Typhimurium mutants with a remodeled outer membrane could induce cross-protection against heteroserotypic Salmonella infection. Therefore, in this study, we further evaluated the potential of broad-spectrum vaccines based on major outer membrane protein (OMP)-deficient OMVs, including ΔompA, ΔompC, and ΔompD, and determined the protection effectiveness of these candidate vaccines in murine and chicken infection models. The results showed that ΔompA led to an increase in the production of OMVs. Notably, ΔompAΔompCΔompD OMVs showed significantly better cross-protection against S. enterica serovar Choleraesuis, S. Enteritidis, APEC O78, and Shigella flexneri 2a than did other omp-deficient OMVs, with the exception of ΔompA OMVs. Subsequently, we verified the results in the chicken model, in which ΔompAΔompCΔompD OMVs elicited significant cross-protection against S. Enteritidis and APEC O78 infections. These findings further confirmed the feasibility of improving the immunogenicity of OMVs by remodeling the outer membrane and provide a new perspective for the development of broad-spectrum vaccines based on OMVs.

scholarly journals Massive Formation of Intracellular Membrane Vesicles in Escherichia coli by a Monotopic Membrane-bound Lipid Glycosyltransferase

2009 ◽  
Vol 284 (49) ◽  
pp. 33904-33914 ◽  
Author(s):  
Hanna M. Eriksson ◽  
Per Wessman ◽  
Changrong Ge ◽  
Katarina Edwards ◽  
Åke Wieslander
Keyword(s):  
Escherichia Coli ◽  
Membrane Vesicles ◽  
Intracellular Membrane ◽  
Membrane Bound

scholarly journals Pmel17 Initiates Premelanosome Morphogenesis within Multivesicular Bodies

2001 ◽  
Vol 12 (11) ◽  
pp. 3451-3464 ◽  
Author(s):  
Joanne F. Berson ◽  
Dawn C. Harper ◽  
Danielle Tenza ◽  
Graça Raposo ◽  
Michael S. Marks
Keyword(s):  
Membrane Protein ◽  
Membrane Vesicles ◽  
Integral Membrane Protein ◽  
Multivesicular Bodies ◽  
Posttranslational Processing ◽  
Tissue Specific ◽  
Physical Linkage ◽  
Golgi Compartment ◽  
Membrane Bound ◽  
Unknown Composition

Melanosomes are tissue-specific organelles within which melanin is synthesized and stored. The melanocyte-specific glycoprotein Pmel17 is enriched in the lumen of premelanosomes, where it associates with characteristic striations of unknown composition upon which melanin is deposited. However, Pmel17 is synthesized as an integral membrane protein. To clarify its physical linkage to premelanosomes, we analyzed the posttranslational processing of human Pmel17 in pigmented and transfected nonpigmented cells. We show that Pmel17 is cleaved in a post-Golgi compartment into two disulfide-linked subunits: a large lumenal subunit, Mα, and an integral membrane subunit, Mβ. The two subunits remain associated intracellularly, indicating that detectable Mα remains membrane bound. We have previously shown that Pmel17 accumulates on intralumenal membrane vesicles and striations of premelanosomes in pigmented cells. In transfected nonpigmented cells Pmel17 associates with the intralumenal membrane vesicles of multivesicular bodies; cells overexpressing Pmel17 also display structures resembling premelanosomal striations within these compartments. These results suggest that Pmel17 is sufficient to drive the formation of striations from within multivesicular bodies and is thus directly involved in the biogenesis of premelanosomes.

scholarly journals Membrane Protein Degradation by FtsH Can Be Initiated from Either End

2002 ◽  
Vol 184 (17) ◽  
pp. 4775-4782 ◽  
Author(s):  
Shinobu Chiba ◽  
Yoshinori Akiyama ◽  
Koreaki Ito
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Exact Sequence ◽  
Integral Membrane Proteins ◽  
Amino Acid Residues ◽  
Aaa Atpase ◽  
Membrane Bound ◽  
Periplasmic Domain

ABSTRACT FtsH, a membrane-bound metalloprotease, with cytoplasmic metalloprotease and AAA ATPase domains, degrades both soluble and integral membrane proteins in Escherichia coli. In this paper we investigated how membrane-embedded substrates are recognized by this enzyme. We showed previously that FtsH can initiate processive proteolysis at an N-terminal cytosolic tail of a membrane protein, by recognizing its length (more than 20 amino acid residues) but not exact sequence. Subsequent proteolysis should involve dislocation of the substrates into the cytosol. We now show that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail and that the initiation efficiency depends on the length of the tail. This mode of degradation also appeared to be processive, which can be aborted by a tightly folded periplasmic domain. These results indicate that FtsH can exhibit processivity against membrane-embedded substrates in either the N-to-C or C-to-N direction. Our results also suggest that some membrane proteins receive bidirectional degradation simultaneously. These results raise intriguing questions about the molecular directionality of the dislocation and proteolysis catalyzed by FtsH.

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