scholarly journals A single amino acid Gly-tag enables metal-free protein purification

2020 ◽  
Vol 11 (48) ◽  
pp. 13137-13142
Author(s):  
Landa Purushottam ◽  
Unnikrishnan V. B. ◽  
Dattatraya Gautam Rawale ◽  
Mansi Gujrati ◽  
Surya Dev Mishra ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Purification ◽  
Single Amino Acid ◽  
Metal Contaminants ◽  
Free Protein ◽  
Metal Free ◽  
N Terminus ◽  
Pure Protein ◽  
User Friendly

Gly-tag resin precisely captures and releases a protein with one glycine at the N-terminus. The user-friendly protocol delivers analytically pure protein free of metal contaminants.

scholarly journals Identification of critical amino acid residues in the regulatory N-terminal domain of PMEL

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Susan M. Mitchell ◽  
Morven Graham ◽  
Xinran Liu ◽  
Ralf M. Leonhardt
Keyword(s):  
Amino Acid ◽  
Pigment Cell ◽  
Specific Protein ◽  
Single Amino Acid ◽  
Amyloid Formation ◽  
Amino Acid Residues ◽  
Proteolytic Fragment ◽  
The Core ◽  
N Terminus ◽  
In Cis

AbstractThe pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked in cis to the CAF in order to drive amyloid formation. The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.

scholarly journals Mutational Analysis of Quinolone Resistance Protein QnrB1

2013 ◽  
Vol 57 (11) ◽  
pp. 5733-5736 ◽  
Author(s):  
George A. Jacoby ◽  
Marian A. Corcoran ◽  
Debra M. Mills ◽  
Caitlin M. Griffin ◽  
David C. Hooper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Essential Amino Acids ◽  
Quinolone Resistance ◽  
Single Amino Acid ◽  
Protective Activity ◽  
N Terminus ◽  
Resistance Protein

ABSTRACTAlanine substitutions and selected deletions have been used to localize amino acids in QnrB essential for its protective activity. Essential amino acids are found at positions i and i−2in the pentapeptide repeat module and in the larger of two loops, where deletion of only a single amino acid compromises activity. Deletion of 10 amino acids at the N terminus is tolerated, but removal of 3 amino acids in the C-terminal dimerization unit destroys activity.

scholarly journals Changing a single amino acid in the N-terminus of murine PrP alters TSE incubation time across three species barriers

2001 ◽  
Vol 20 (18) ◽  
pp. 5070-5078 ◽  
Author(s):  
Rona M. Barron ◽  
Val Thomson ◽  
Elizabeth Jamieson ◽  
David W. Melton ◽  
James Ironside ◽  
...  
Keyword(s):  
Amino Acid ◽  
Incubation Time ◽  
Single Amino Acid ◽  
N Terminus

scholarly journals INDUCTION OF RABBIT ANTIBODY WITH MOLECULAR UNIFORMITY AFTER IMMUNIZATION WITH GROUP C STREPTOCOCCI

1970 ◽  
Vol 131 (1) ◽  
pp. 207-221 ◽  
Author(s):  
Klaus Eichmann ◽  
Henry Lackland ◽  
Leroy Hood ◽  
Richard M. Krause
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Variable Region ◽  
Disc Electrophoresis ◽  
Single Amino Acid ◽  
Antigenic Specificity ◽  
Light Chains ◽  
Rabbit Antibody ◽  
N Terminus

Antibodies with uniform properties may occur in rabbits after immunization with Group C streptococci. These precipitating antibodies possess specificity for the group-specific carbohydrate. Not uncommonly, their concentration is between 20 and 40 mg/ml of antiserum. Evidence for molecular uniformity in the case of one of these antibodies, described in detail here, includes: individual antigenic specificity; monodisperse distribution of the light chains by alkaline urea polyacrylamide disc electrophoresis; and a single amino acid in each of the first three N-terminal positions of the light chains. When the amino acid sequence of rabbit antibody b+ light chains (κ type) are aligned against their human κ counterparts, a definite homology is observed between the N-terminus of the human and the rabbit variable region.

Single-site labeling of histidine in proteins, on-demand reversibility, and traceless metal-free protein purification

2019 ◽  
Vol 55 (8) ◽  
pp. 1100-1103 ◽  
Author(s):  
Pralhad Namdev Joshi ◽  
Vishal Rai
Keyword(s):  
Protein Purification ◽  
Single Site ◽  
Free Protein ◽  
Metal Free ◽  
On Demand ◽  
Multiple Copies

A precision methodology distinguishes one His from all the nucleophilic residues and its multiple copies.

scholarly journals Roles of Specific Amino Acids in the N Terminus of Pseudomonas aeruginosa Flagellin and of Flagellin Glycosylation in the Innate Immune Response

2005 ◽  
Vol 73 (12) ◽  
pp. 8237-8246 ◽  
Author(s):  
Amrisha Verma ◽  
Shiwani K. Arora ◽  
Sudha K. Kuravi ◽  
Reuben Ramphal
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Binding Site ◽  
A549 Cells ◽  
Single Amino Acid ◽  
Wild Type ◽  
Binding Region ◽  
N Terminus ◽  
Flagellin Glycosylation

ABSTRACT The Toll-like receptor 5 (TLR5) binding site has been predicted to be in the N terminus of the flagellin molecule. In order to better define the interaction between the N-terminal amino acids of Pseudomonas aeruginosa flagellin and TLR5, site-specific mutations were generated between residues 88 and 97 of P. aeruginosa PAK flagellin as well as outside of this region. The mutant flagellins were expressed in Escherichia coli BL21(plysS), purified by affinity chromatography, and passed through a polymyxin B column to remove contaminating lipopolysaccharide (LPS). Their ability to stimulate interleukin-8 (IL-8) release from A549 cells was examined. The cloned mutated genes were used to complement a PAK fliC mutant in order to test for effects on motility and on IL-8 release by purified flagellar preparations. All the mutations, single or double, in the predicted TLR5 binding region reduced IL-8 signaling to less than 95% of the wild-type flagellin levels, but the single mutation outside the binding region had no effect. Changes made at two amino acid sites resulted in loss/reduction of motility; however, changes made at single sites, i.e., Q83A, L88A, R90A, M91A, L94A, and Q97A, had no effect on motility. The mutated genes encoding two of the motile but poorly signaling flagellins had no compensatory mutations to allow motility. Thus, while it is speculated that pathogen-associated molecular patterns (PAMPs) have evolved in locations that are essential to maintain function, it appears that there is tolerance for at least single amino acid changes in the PAMP of P. aeruginosa flagellin. The purpose of flagellin glycosylation in P. aeruginosa is unknown. In order to examine its role, if any, in signaling an inflammatory response, we used whole flagella from the motile chromosomal mutant strains PAKrfbC and PAO1rfbC, which are defective in flagellin glycosylation. IL-8 release from A549 cells stimulated with nonglycosylated flagellar preparations (having less then 1 picogram of LPS/μg) was significantly reduced compared to their respective wild-type flagellar preparations, indicating a role of flagellar glycosylation in the proinflammatory action of Pseudomonas flagellin. The basis of the latter activity is unknown, since the glycosylation sites are found in the D3 domain of flagellins and the TLR5 binding site is located in the D1 domain. Thus, P. aeruginosa flagellin has evolved additional flagellar signaling mechanisms over that described for Salmonella flagellin.

Single Amino Acid Substitutions at the N-Terminus of a Recombinant Cytotoxic Ribonuclease Markedly Influence Biochemical and Biological Properties†

Biochemistry ◽  
1998 ◽  
Vol 37 (15) ◽  
pp. 5173-5183 ◽  
Author(s):  
Dianne L. Newton ◽  
Lluis Boque ◽  
Alexander Wlodawer ◽  
Charles Y. Huang ◽  
Susanna M. Rybak
Keyword(s):  
Amino Acid ◽  
Biological Properties ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
N Terminus
Sign in / Sign up

Export Citation Format

Share Document