scholarly journals Genetically Encoded Tetrazine Amino Acid Directs Rapid Site-Specific in Vivo Bioorthogonal Ligation with trans-Cyclooctenes

2012 ◽  
Vol 134 (6) ◽  
pp. 2898-2901 ◽  
Author(s):  
Jason L. Seitchik ◽  
Jennifer C. Peeler ◽  
Michael T. Taylor ◽  
Melissa L. Blackman ◽  
Timothy W. Rhoads ◽  
...  
Keyword(s):  
Amino Acid ◽  
Site Specific

Site Specific Incorporation of Amino Acid Analogues into Proteins In Vivo

2000 ◽  
Author(s):  
Anne K. Kowal ◽  
Caroline Kohrer ◽  
Uttam L. RajBhandary
Keyword(s):  
Amino Acid ◽  
Site Specific ◽  
Amino Acid Analogues

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...

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

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.

Site-Specific 89Zr- and 111In-Radiolabeling and In Vivo Evaluation of Glycan-free Antibodies by Azide–Alkyne Cycloaddition with a Non-natural Amino Acid

2020 ◽  
Vol 31 (4) ◽  
pp. 1177-1187 ◽  
Author(s):  
Shin Hye Ahn ◽  
Brett A. Vaughn ◽  
Willy A. Solis ◽  
Mark L. Lupher ◽  
Trevor J. Hallam ◽  
...  
Keyword(s):  
Amino Acid ◽  
Natural Amino Acid ◽  
In Vivo Evaluation ◽  
Site Specific
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