Secretory expression and surface display of a new and biologically active single-chain insulin (SCI-59) analog by lactic acid bacteria

2017 ◽  
Vol 101 (8) ◽  
pp. 3259-3271 ◽  
Author(s):  
Ruifeng Mao ◽  
Dongli Wu ◽  
Shimeng Hu ◽  
Kangping Zhou ◽  
Man Wang ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Surface Display ◽  
Biologically Active ◽  
Single Chain ◽  
Secretory Expression ◽  
Single Chain Insulin

scholarly journals A SH3_5 Cell Anchoring Domain for Non-recombinant Surface Display on Lactic Acid Bacteria

2021 ◽  
Vol 8 ◽  
Author(s):  
Pei Kun Richie Tay ◽  
Pei Yu Lim ◽  
Dave Siak-Wei Ow
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Surface Display ◽  
Bioactive Molecules ◽  
Gastric Digestion ◽  
Functional Protein ◽  
Fluorescent Reporter ◽  
Surface Conditions ◽  
Bacterial Surface

Lactic acid bacteria (LAB) are a group of gut commensals increasingly recognized for their potential to deliver bioactive molecules in vivo. The delivery of therapeutic proteins, in particular, can be achieved by anchoring them to the bacterial surface, and various anchoring domains have been described for this application. Here, we investigated a new cell anchoring domain (CAD4a) isolated from a Lactobacillus protein, containing repeats of a SH3_5 motif that binds non-covalently to peptidoglycan in the LAB cell wall. Using a fluorescent reporter, we showed that C-terminal CAD4a bound Lactobacillus fermentum selectively out of a panel of LAB strains, and cell anchoring was uniform across the cell surface. Conditions affecting CAD4a anchoring were studied, including temperature, pH, salt concentration, and bacterial growth phase. Quantitative analysis showed that CAD4a allowed display of 105 molecules of monomeric protein per cell. We demonstrated the surface display of a functional protein with superoxide dismutase (SOD), an antioxidant enzyme potentially useful for treating gut inflammation. SOD displayed on cells could be protected from gastric digestion using a polymer matrix. Taken together, our results show the feasibility of using CAD4a as a novel cell anchor for protein surface display on LAB.

scholarly journals Co-Microencapsulation of Anthocyanins from Black Currant Extract and Lactic Acid Bacteria in Biopolymeric Matrices

Molecules ◽  
2020 ◽  
Vol 25 (7) ◽  
pp. 1700 ◽  
Author(s):  
Iuliana Maria Enache ◽  
Aida Mihaela Vasile ◽  
Elena Enachi ◽  
Vasilica Barbu ◽  
Nicoleta Stănciuc ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Biological Activities ◽  
Biologically Active Compounds ◽  
Biologically Active ◽  
Confocal Laser ◽  
In Vitro Digestibility ◽  
Black Currant ◽  
Active Compounds ◽  
Food Ingredients

Anthocyanins from black currant extract and lactic acid bacteria were co-microencapsulated using a gastro-intestinal-resistant biocomposite of whey protein isolate, inulin, and chitosan, with an encapsulation efficiency of 95.46% ± 1.30% and 87.38% ± 0.48%, respectively. The applied freeze-drying allowed a dark purple stable powder to be obtained, with a satisfactory content of phytochemicals and 11 log colony forming units (CFU)/g dry weight of powder (DW). Confocal laser microscopy displayed a complex system, with several large formations and smaller aggregates inside, consisting of biologically active compounds, lactic acid bacteria cells, and biopolymers. The powder showed good storage stability, with no significant changes in phytochemicals and viable cells over 3 months. An antioxidant activity of 63.64 ± 0.75 mMol Trolox/g DW and an inhibitory effect on α-amylase and α-glucosidase of 87.10% ± 2.08% and 36.96% ± 3.98%, respectively, highlighted the potential biological activities of the co-microencapsulated powder. Significantly, the in vitro digestibility profile showed remarkable protection in the gastric environment, with controlled release in the intestinal simulated environment. The powder was tested by addition into a complex food matrix (yogurt), and the results showed satisfactory stability of biologically active compounds when stored for 21 d at 4 °C. The obtained results confirm the important role of microencapsulation in ensuring a high degree of protection, thus allowing new approaches in developing food ingredients and nutraceuticals, with enhanced functionalities.

scholarly journals Characterization of a Novel LysM Domain from Lactobacillus fermentum Bacteriophage Endolysin and Its Use as an Anchor To Display Heterologous Proteins on the Surfaces of Lactic Acid Bacteria

2010 ◽  
Vol 76 (8) ◽  
pp. 2410-2418 ◽  
Author(s):  
Shumin Hu ◽  
Jian Kong ◽  
Wentao Kong ◽  
Tingting Guo ◽  
Mingjie Ji
Keyword(s):  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Fluorescent Protein ◽  
Binding Capacity ◽  
Surface Display ◽  
Lactobacillus Fermentum ◽  
Display System ◽  
C Terminus ◽  
Bacteriophage Endolysin

ABSTRACT The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage φPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli. After being mixed with various cells in vitro, GFP was successfully displayed on the surfaces of Lactococcus lactis, Lactobacillus casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, β-galactosidase (β-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and β-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB.

scholarly journals Adjuvant Effects for Oral Immunization Provided by Recombinant Lactobacillus casei Secreting Biologically Active Murine Interleukin-1β

2009 ◽  
Vol 17 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Akinobu Kajikawa ◽  
Kazuya Masuda ◽  
Mitsunori Katoh ◽  
Shizunobu Igimi
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactobacillus Casei ◽  
Oral Immunization ◽  
Biologically Active ◽  
Interleukin 1Β ◽  
Intestinal Loop ◽  
Adjuvant Effects

ABSTRACT Vaccine delivery systems using lactic acid bacteria are under development, but their efficiency is insufficient. Autologous cytokines, such as interleukin-1β (IL-1β), are potential adjuvants for mucosal vaccines and can be provided by recombinant lactic acid bacteria. The aim of this study was the construction and evaluation of recombinant Lactobacillus casei producing IL-1β as an adjuvant delivery agent. The recombinant strain was constructed using an expression/secretion vector plasmid, including a mature IL-1β gene from mouse. The biological activity of the cytokine was confirmed by IL-8 production from Caco-2 cells. In response to the recombinant L. casei secreting IL-1β, expression of IL-6 was detected in vivo using a ligated-intestinal-loop assay. The release of IL-6 from Peyer's patch cells was also detected in vitro. Intragastric immunization with heat-killed Salmonella enterica serovar Enteritidis (SE) in combination with IL-1β-secreting lactobacilli resulted in relatively high SE-specific antibody production. In this study, it was demonstrated that recombinant L. casei secreting bioactive murine IL-1β provided adjuvant effects for intragastric immunization.

scholarly journals Lactic Acid Bacteria as a Surface Display Platform for Campylobacter jejuni Antigens

2015 ◽  
Vol 25 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Patrycja Kobierecka ◽  
Agnieszka Wyszyńska ◽  
Marta Maruszewska ◽  
Anna Wojtania ◽  
Joanna Żylińska ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Campylobacter Jejuni ◽  
Surface Display ◽  
Food Poisoning ◽  
Lactobacillus Salivarius ◽  
Binding Ability ◽  
Hybrid Proteins ◽  
Mucosal Delivery ◽  
Delivery Vehicles

<b><i>Background:</i></b> Food poisoning and diarrheal diseases continue to pose serious health care and socioeconomic problems worldwide. <i>Campylobacter</i> spp. is a very widespread cause of gastroenteritis. Over the past decade there has been increasing interest in the use of lactic acid bacteria (LAB) as mucosal delivery vehicles. They represent an attractive opportunity for vaccination in addition to vaccination with attenuated bacterial pathogens. <b><i>Methods:</i></b> We examined the binding ability of hybrid proteins to nontreated or trichloroacetic acid (TCA)-pretreated LAB cells by immunofluorescence and Western blot analysis. <b><i>Results:</i></b> In this study we evaluated the possibility of using GEM (Gram-positive enhancer matrix) particles of <i>Lactobacillus salivarius</i> as a binding platform for 2 conserved, immunodominant, extracytoplasmic <i>Campylobacter jejuni</i> proteins: CjaA and CjaD. We analyzed the binding ability of recombinant proteins that contain <i>C. jejuni</i> antigens (CjaA or CjaD) fused with the protein anchor (PA) of the <i>L. lactis </i>peptidoglycan hydrolase AcmA, which comprises 3 LysM motifs and determines noncovalent binding to the cell wall peptidoglycan. Both fused proteins, i.e. 6HisxCjaAx3LysM and 6HisxCjaDx3LysM, were able to bind to nontreated or TCA-pretreated <i>L. salivarius</i> cells. <b><i>Conclusion:</i></b> Our results documented that the LysM-mediated binding system allows us to construct GEM particles that present 2 <i>C. jejuni</i> antigens.

scholarly journals Heterologous surface display on lactic acid bacteria: non-GMO alternative?

Bioengineered ◽  
2015 ◽  
Vol 6 (3) ◽  
pp. 179-183 ◽  
Author(s):  
Petra Zadravec ◽  
Borut Štrukelj ◽  
Aleš Berlec
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Surface Display

Review: Bacteriocins of Lactic Acid Bacteria

2001 ◽  
Vol 7 (4) ◽  
pp. 281-305 ◽  
Author(s):  
L. M. Cintas ◽  
M. P. Casaus ◽  
C. Herranz ◽  
I. F. Nes ◽  
P. E. Hernández
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Secretory Pathway ◽  
Response Regulator ◽  
Ionic Channels ◽  
Class Ii ◽  
Biologically Active ◽  
Leader Peptide ◽  
Class Iii ◽  
Activity Spectrum

During the last few years, a large number of new bacteriocins produced by lactic acid bacteria (LAB) have been identified and characterized. LAB-bacteriocins comprise a heterogeneous group of physicochemically diverse ribosomally-synthesized peptides or proteins showing a narrow or broad antimicrobial activity spectrum against Gram-positive bacteria. Bacteriocins are classified into separate groups such as the lantibiotics (Class I); the small (<10 kDa) heat-stable postranslationally unmodified non-lantibiotics (Class II), further subdivided in the pediocin-like and anti Listeria bacteriocins (subclass IIa), the two-peptide bacteriocins (subclass IIb), and the sec-dependent bacteriocins (subclass IIc); and the large (>30 kDa) heat-labile non-lantibiotics (Class III). Most bacteriocins characterized to date belong to Class II and are synthesized as precursor peptides (preprobacteriocins) containing an N-terminal double-glycine leader peptide, which is cleaved off concomitantly with externalization of biologically active bacteriocins by a dedicated ABC-transporter and its accessory protein. However, the recently identified sec-dependent bacteriocins contain an N-terminal signal peptide that directs bacteriocin secretion through the general secretory pathway (GSP). Most LAB-bacteriocins act on sensitive cells by destabilization and permeabilization of the cytoplasmic membrane through the formation of transitory poration complexes or ionic channels that cause the reduction or dissipation of the proton motive force (PMF). Bacteriocin producing LAB strains protect themselves against the toxicity of their own bacteriocins by the expression of a specific immunity protein which is generally encoded in the bacteriocin operon. Bacteriocin production in LAB is frequently regulated by a three-component signal transduction system consisting of an induction factor (IF), and histidine protein kinase (HPK) and a response regulator (RR). This paper presents an updated review on the general knowledge about physicochemical properties, molecular mode of action, biosynthesis, regulation and genetics of LAB-bacteriocins.

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