scholarly journals Determination of the Domain of the Lactobacillus delbrueckii subsp. bulgaricus Cell Surface Proteinase PrtB Involved in Attachment to the Cell Wall after Heterologous Expression of the prtB Gene in Lactococcus lactis

2003 ◽  
Vol 69 (6) ◽  
pp. 3377-3384 ◽  
Author(s):  
Jacques-Edouard Germond ◽  
Mich�le Delley ◽  
Christophe Gilbert ◽  
Dani�le Atlan
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Cell Surface ◽  
Lactococcus Lactis ◽  
Culture Medium ◽  
Surface Display ◽  
Cell Surface Display ◽  
Lactobacillus Delbrueckii ◽  
Heterologous Proteins

ABSTRACT Belonging to the subtilase family, the cell surface proteinase (CSP) PrtB of Lactobacillus delbrueckii subsp. bulgaricus differs from other CSPs synthesized by lactic acid bacteria. Expression of the prtB gene under its own promoter was shown to complement the proteinase-deficient strain MG1363 (PrtP− PrtM−) of Lactococcus lactis subsp. cremoris. Surprisingly, the maturation process of PrtB, unlike that of lactococcal CSP PrtPs, does not require a specific PrtM-like chaperone. The carboxy end of PrtB was previously shown to be different from the consensus anchoring region of other CSPs and exhibits an imperfect duplication of 59 amino acids with a high lysine content. By using a deletion strategy, the removal of the last 99 amino acids, including the degenerated anchoring signal (LPKKT), was found to be sufficient to release a part of the truncated PrtB into the culture medium and led to an increase in PrtB activity. This truncated PrtB is still active and enables L. lactis MG1363 to grow in milk supplemented with glucose. By contrast, deletion of the last 806 amino acids of PrtB led to the secretion of an inactive proteinase. Thus, the utmost carboxy end of PrtB is involved in attachment to the bacterial cell wall. Proteinase PrtB constitutes a powerful tool for cell surface display of heterologous proteins like antigens.

Construction of a novel system for cell surface display of heterologous proteins on Pichia pastoris

2007 ◽  
Vol 29 (10) ◽  
pp. 1561-1566 ◽  
Author(s):  
Qingjie Wang ◽  
Lei Li ◽  
Min Chen ◽  
Qingsheng Qi ◽  
Peng George Wang
Keyword(s):  
Pichia Pastoris ◽  
Cell Surface ◽  
Surface Display ◽  
Cell Surface Display ◽  
Heterologous Proteins

scholarly journals Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

2002 ◽  
Vol 68 (6) ◽  
pp. 2917-2923 ◽  
Author(s):  
Eric Bernasconi ◽  
Jacques-Edouard Germond ◽  
Michèle Delley ◽  
Rodolphe Fritsché ◽  
Blaise Corthésy
Keyword(s):  
Cell Surface ◽  
Lactococcus Lactis ◽  
Fusion Proteins ◽  
Surface Display ◽  
Major Allergen ◽  
Full Length ◽  
Lactobacillus Bulgaricus ◽  
Heterologous Proteins ◽  
Food Antigens ◽  
Β Lactoglobulin

ABSTRACT Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB∂, PrtB∂-BLG, and PrtB∂-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB∂-BLG yielded up to 170 μg per 109 CFU in the culture supernatant and 9 μg per 109 CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.

scholarly journals Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response

2006 ◽  
Vol 72 (11) ◽  
pp. 7140-7147 ◽  
Author(s):  
Frank Breinig ◽  
Björn Diehl ◽  
Sabrina Rau ◽  
Christian Zimmer ◽  
Helmut Schwab ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Cell Surface ◽  
Surface Display ◽  
Cell Surface Display ◽  
Constitutive Activation ◽  
Unfolded Protein ◽  
Yeast Cell Surface ◽  
Protein Response

ABSTRACT Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg−1 protein for Kre1/EstA/Cwp2p and 72 mU mg−1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg−1 protein for Kre1/EstA/Cwp2p and 1.27 U mg−1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

Efficient cell surface display of Lip2 lipase using C-domains of glycosylphosphatidylinositol-anchored cell wall proteins of Yarrowia lipolytica

2011 ◽  
Vol 91 (3) ◽  
pp. 645-654 ◽  
Author(s):  
Evgeniya Y. Yuzbasheva ◽  
Tigran V. Yuzbashev ◽  
Ivan A. Laptev ◽  
Tatiana K. Konstantinova ◽  
Sergey P. Sineoky
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Yarrowia Lipolytica ◽  
Surface Display ◽  
Cell Surface Display ◽  
Cell Wall Proteins ◽  
Lip2 Lipase

scholarly journals Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids

2006 ◽  
Vol 103 (27) ◽  
pp. 10180-10185 ◽  
Author(s):  
A. J. Link ◽  
M. K. S. Vink ◽  
N. J. Agard ◽  
J. A. Prescher ◽  
C. R. Bertozzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Surface Display ◽  
Cell Surface Display ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Aminoacyl Trna Synthetase ◽  
Noncanonical Amino Acids

scholarly journals Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL

2012 ◽  
Vol 78 (9) ◽  
pp. 3051-3058 ◽  
Author(s):  
Hyeok-Jin Ko ◽  
Eunhye Park ◽  
Joseph Song ◽  
Taek Ho Yang ◽  
Hee Jong Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Surface Display ◽  
Cell Surface Display ◽  
Heterologous Proteins ◽  
Display System ◽  
Reporter Protein ◽  
Content Type ◽  
Ligation Independent Cloning

ABSTRACTAutotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) ofEscherichia colifor the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes fromSaccharophagus degradans2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 104molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growingE. coli.

Cloning, molecular characterization, and expression of the genes encoding the lytic functions of lactococcal bacteriophage ϕLCE: a dual lysis system of modular design

1994 ◽  
Vol 40 (8) ◽  
pp. 658-665 ◽  
Author(s):  
Nils-Kåre Birkeland
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Lactococcus Lactis ◽  
Modular Design ◽  
Sequence Similarity ◽  
Lactobacillus Delbrueckii ◽  
Host Cells ◽  
Lytic Enzyme ◽  
E Coli ◽  
Genes Encoding

The genes encoding the lysis proteins of Lactococcus lactis bacteriophage [Formula: see text] were cloned, sequenced, and expressed in Escherichia coli. The [Formula: see text] lysis genes, lysA and lysB, encode a membrane-disrupting protein (LysA) of 88 amino acids, and a cell wall degrading protein (LysB) of 429 amino acids, which shares significant sequence similarity with lysins from the Streptococcus pneumoniae phages Cp-1, Cp-7, and Cp-9, and Lactobacillus delbrueckii phage mv 1. Both LysA and LysB function in E. coli, as judged by lysis of the E. coli host cells and by lytic activity against lactococcal cells when the cloned lysA and lysB genes are expressed. The LysA protein possesses two putative transmembrane helices and highly charged N- and C-termini, and is structurally similar to phage holins that are known to induce lesions in the inner membrane through which phage endolysin can be released to its cell wall substrate. The C-terminal end of LysB contains two highly homologous sequence repeats of 43 amino acids. The LysB repeats show strong sequence similarity to repeats found in lytic enzymes from other Gram-positive bacteria and from Bacillus subtilis phage [Formula: see text] and PZA, as well as in some functionally unrelated proteins, and they are possibly involved in binding of the enzyme to the cell wall substrate. The organization of the dual [Formula: see text] lysis system supports earlier suggestions that exchange of modular units is an important principle in protein evolution.Key words: amino acid sequence repeats, bacteriophage [Formula: see text], holin, Lactococcus lactis, lysin, lytic enzyme.

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