Site Specific Combinations of Stabilizing and Destabilizing Amino Acid Replacements in Yeast Cytochrome c: In Vivo and in Vitro Effects

Biochemistry ◽  
1995 ◽  
Vol 34 (21) ◽  
pp. 7103-7112 ◽  
Author(s):  
Lisa I. Linske-O'Connell ◽  
Fred Sherman ◽  
George McLendon
Keyword(s):  
Amino Acid ◽  
Cytochrome C ◽  
Site Specific ◽  
In Vitro Effects

Stabilizing Amino Acid Replacements at Position 52 in Yeast Iso-1-cytochrome c: In Vivo and in Vitro Effects

Biochemistry ◽  
1995 ◽  
Vol 34 (21) ◽  
pp. 7094-7102 ◽  
Author(s):  
Lisa I. Linske-O'Connell ◽  
Fred Sherman ◽  
George McLendon
Keyword(s):  
Amino Acid ◽  
Cytochrome C ◽  
In Vitro Effects

Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

2021 ◽  
Author(s):  
Babu Sudhamalla ◽  
Anirban Roy ◽  
Soumen Barman ◽  
Jyotirmayee Padhan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Unnatural Amino Acids ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

scholarly journals Interaction of the FimB Integrase with thefimS Invertible DNA Element in Escherichia coliIn Vivo and In Vitro

2000 ◽  
Vol 182 (10) ◽  
pp. 2953-2959 ◽  
Author(s):  
Lesley S. Burns ◽  
Stephen G. J. Smith ◽  
Charles J. Dorman
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence Analysis ◽  
Specific Component ◽  
Genetic Switch ◽  
Site Specific ◽  
Tyrosine Recombinases ◽  
Catalytic Motif ◽  
A Site

ABSTRACT The FimB protein is a site-specific recombinase that inverts thefimS genetic switch in Escherichia coli. Based on amino acid sequence analysis alone, FimB has been assigned to the integrase family of tyrosine recombinases. We show that amino acid substitutions at positions R47, H141, R144, and Y176, corresponding to highly conserved members of the catalytic motif of integrase proteins, render FimB incapable of inverting the fimS element in vivo. The arginine substitutions reduced the ability of FimB to bind tofimS in vivo or in vitro, while the substitution R144Q resulted in a protein unable to bind independently to the half sites located at the left end of fimS in phase-on bacteria. These data confirm that FimB is an integrase and suggest that residue R144 has a role in binding to a specific component of the fimswitch.

scholarly journals SAT-283 Generation of a Long-Acting Human Growth Hormone Receptor Antagonist by Site-Specific Pegylation

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Yue Wang ◽  
Ries J Langley ◽  
Kyle Tamshen ◽  
Heather D Maynard ◽  
Stephen M F Jamieson ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Acid Site ◽  
Half Life ◽  
In Vitro Bioactivity ◽  
Site Specific ◽  
Sds Page ◽  
Long Acting

Abstract Growth hormone (GH) is a peptide hormone that mediates actions through binding to a cell surface GH receptor (GHR), activating key signalling pathways including the JAK/STAT pathway. Excess GH secretion leads to acromegaly and tumoral expression has been implicated in cancer progression, suggesting that GH is also a potential target for anticancer therapy. Pegvisomant is the only GHR antagonist approved for clinical use. This antagonist is a PEGylated form of a mutated GH (B2036) that binds and blocks the receptor. Conjugation to polyethylene glycol (PEG) at multiple amine residues reduces in vitro bioactivity but extends the serum half-life resulting in improved in vivo bioactivity. We investigated whether we could generate a long-acting PEGylated GHR antagonist through site-specific conjugation of PEG. A codon optimised GHR antagonist, with an introduced free cysteine residue at amino acid site 144 (S144C), was generated by gene synthesis and recombinantly engineered by gene fusion with thioredoxin. Recombinant protein was expressed in E. coli and purified using a series of chromatographic methods. Antagonists were PEGylated using cysteine-specific conjugation chemistry. In vitro activity was determined using a Ba/F3-GHR viability assay, and in vivo pharmacokinetic and bioactivity was determined in mice. Fusion to thioredoxin was found to improve soluble protein expression at 30℃, resulting in dramatically increased yield. After a series of purification steps, including Ni-NTA, 3C protease cleavage and ion-exchange chromatography, a single band with a molecular mass of 22 kDa was observed by SDS-PAGE analysis. The recombinant antagonist was conjugated to 20 kDa or 30 kDa-PEG at amino acid site S144C. After purification, a single band with an effective molecular size of approximately 60 kDa (PEG-20kDa conjugate) or 70 kDa (PEG-30kDa conjugate) was observed by SDS-PAGE analysis. The unconjugated antagonist inhibited the proliferation of Ba/F3-GHR cells in a dose-dependent manner with a half maximal inhibitory concentration (IC50) of 10.1 ± 2.5 nM. Following PEGylation and purification, the PEG-20kDa and PEG-30kDa conjugates retained high in vitro bioactivity with an IC50 of 66.2 ± 3.8 nM and 106.1 ± 7.1 nM, respectively. Pharmacokinetic analysis demonstrated that PEGylation increased the serum half-life to approximately 15 hours in mice. Subcutaneous administration of the PEG-30kDa conjugate (10 mg/kg/day) reduced serum IGF-I levels in mice. In conclusion, we have generated a novel long-acting human GHR antagonist conjugate by introducing a free cysteine at a non-essential site of the antagonist and targeted attachment of PEG.

scholarly journals Discordant Effects of Putative Lysine Acetyltransferase Inhibitors in Biochemical and Living Systems

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1022
Author(s):  
Henry ◽  
Kuo ◽  
Siegel ◽  
Yen ◽  
Rhodes ◽  
...  
Keyword(s):  
Enzyme Inhibitors ◽  
Model Systems ◽  
Zebrafish Model ◽  
Site Specific ◽  
Starting Point ◽  
Lysine Residues ◽  
Site Targeting ◽  
In Vitro Effects

Lysine acetyltransferases (KATs) are exquisitely fine-tuned to target specific lysine residues on many proteins, including histones, with aberrant acetylation at distinct lysines implicated in different pathologies. However, researchers face a lack of molecular tools to probe the importance of site-specific acetylation events in vivo. Because of this, there can be a disconnect between the predicted in silico or in vitro effects of a drug and the actual observable in vivo response. We have previously reported on how an in vitro biochemical analysis of the site-specific effects of the compound C646 in combination with the KAT p300 can accurately predict changes in histone acetylation induced by the same compound in cells. Here, we build on this effort by further analyzing a number of reported p300 modulators, while also extending the analysis to correlate the effects of these drugs to developmental and phenotypical changes, utilizing cellular and zebrafish model systems. While this study demonstrates the utility of biochemical models as a starting point for predicting in vivo activity of multi-site targeting KATs, it also highlights the need for the development of new enzyme inhibitors that are more specific to the regulation of KAT activity in vivo.

In vivo and in vitro effects of staphylococcal alpha-toxin on amino acid activation in the aorta

1976 ◽  
Vol 25 (15) ◽  
pp. 1733-1736
Author(s):  
J. Patelski ◽  
B. Zgorzalewicz ◽  
S. Szulic ◽  
Z. Waligóra ◽  
J. Jeljaszewicz
Keyword(s):  
Amino Acid ◽  
Acid Activation ◽  
Alpha Toxin ◽  
Amino Acid Activation ◽  
In Vitro Effects

Anti-obesity role of Aster glehni extract: in vivo and in vitro effects

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
HM Lee ◽  
TG Ahn ◽  
CW Kim ◽  
HJ An
Keyword(s):  
In Vitro Effects

Comparison of Specific Antibody, D-Phe-Pro-Arg-CH2Cl and Aprotinin for Prevention of In Vitro Effects of Recombinant Tissue-Type Plasminogen Activator on Haemostasis Parameters

1987 ◽  
Vol 58 (03) ◽  
pp. 921-926 ◽  
Author(s):  
E Seifried ◽  
P Tanswell
Keyword(s):  
Specific Antibody ◽  
Plasma Concentrations ◽  
Fibrinolytic Therapy ◽  
Tissue Type ◽  
Control Value ◽  
Type Plasminogen Activator ◽  
In Vitro Effects ◽  
Fibrinolytic Parameters

SummaryIn vitro, concentration-dependent effects of rt-PA on a range of coagulation and fibrinolytic assays in thawed plasma samples were investigated. In absence of a fibrinolytic inhibitor, 2 μg rt-PA/ml blood (3.4 μg/ml plasma) caused prolongation of clotting time assays and decreases of plasminogen (to 44% of the control value), fibrinogen (to 27%), α2-antiplasmin (to 5%), FV (to 67%), FVIII (to 41%) and FXIII (to 16%).Of three inhibitors tested, a specific polyclonal anti-rt-PA antibody prevented interferences in all fibrinolytic and most clotting assays. D-Phe-Pro-Arg-CH2Cl (PPACK) enabled correct assays of fibrinogen and fibrinolytic parameters but interfered with coagulometric assays dependent on endogenous thrombin generation. Aprotinin was suitable only for a restricted range of both assay types.Most in vitro effects were observed only with rt-PA plasma concentrations in excess of therapeutic values. Nevertheless it is concluded that for clinical application, collection of blood samples on either specific antibody or PPACK is essential for a correct assessment of in vivo effects of rt-PA on the haemostatic system in patients undergoing fibrinolytic therapy.

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