PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology
Abstract
:1. Introduction
2. Results
2.1. Cloning and Bacterial Production of PASylated Tα1
2.2. Purification and In Vitro Characterization of PASylated Tα1
2.3. PASylation Strongly Prolongs Tα1 Pharmacokinetics in Rats
3. Discussion
4. Materials and Methods
4.1. Construction of the Expression Plasmid
4.2. Bacterial Production of Recombinant PASylated Tα1
4.3. Purification of PASylated Tα1
4.4. Analytical Size Exclusion Chromatography
4.5. Endotoxin Quantification
4.6. Western Blot Analysis
4.7. Reverse-Phase Chromatography (RPC) and ESI Mass Spectrometry
4.8. Pharmacokinetic Analysis in Rats
4.9. Quantification of Purified Tα1-PAS
4.10. ELISA Quantification of PASylated Tα1
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AEX | Anion exchange |
AP | Alkaline phosphatase |
aTc | Anhydrotetracycline hydrochloride |
AUC | Area und the curve |
BCIP | 5-Bromo-4-chloro-3-indoxyl phosphate |
BSA | Bovine serum albumin |
CEX | Cation exchange |
CL | Clearance |
Cmax | Maximum (or peak) serum concentration |
CNP | C-type natriuretic peptide |
CORYNEX | Corynebacterium glutamicum protein expression system |
COVID-19 | Severe acute respiratory syndrome coronavirus 2 |
CTLA-4 | Cytotoxic T-lymphocyte-associated protein 4 |
CV | Column volume |
DCs | Dendritic cells |
DPP-IV | Dipeptidyl peptidase-4 |
ELISA | Enzyme-linked immunosorbent assay |
ESETEC | E. coli secretion technology |
ESI-MS | Electrospray ionization mass spectrometry |
FPLC | Fast protein liquid chromatography |
GCC | Glucagon |
GIP | Glucose-dependent insulinotropic polypeptide |
GLP-1 | Glucagon-like peptide 1 |
GM-CSF | Granulocyte-macrophage colony-stimulating factor |
GSH | γ-L-Glutamyl-L-cysteinyl-glycine |
HBV | Hepatitis B virus |
HCV | Hepatitis C virus |
hGH | Human growth hormone |
hIFN-α | Human interferon 1 |
HIV-1 | Human immunodeficiency virus I |
iRGD | Internalizing arginylglycylaspartic acid peptide |
K3-EDTA | Tri-potassium ethylenediaminetetraacetic acid |
MHC | Major histocompatibility complex |
MOPS | 3-(N-morpholino)propanesulfonic acid |
NBT | Nitro blue tetrazolium |
OD | Optical density at 550 nm |
PAS | Proline/alanine-rich sequence |
PAS-Tα1 | N-terminally PASylated Tα1 |
PBS | Phosphate-buffered saline |
PD | Pharmacodynamics |
PEG | Polyethylene glycol |
p.i. | Post injection |
PK | Pharmacokinetics |
ProTα | Prothymosin alpha |
PTH | Parathyroid hormone |
Q-TOF | Quadrupole time of flight |
RBS | Ribosome-binding site |
RGD | Arginylglycylaspartic acid |
RimJ | Ribosomal-protein-S5-alanine N-acetyltransferase |
RPC | Reverse phase chromatography |
RT | Room temperature |
SEC | Size exclusion chromatography |
SDS-PAGE | Sodium dodecyl sulfate polyacrylamide gel electrophoresis |
τ1/2α | Half-life of distribution |
τ1/2β | Elimination half-life |
Tα1 | Thymosin alpha 1 |
Tα1-PAS | C-terminally PASylated Thymosin alpha 1 |
tetp/o | Tetracycline promoter/operator |
Th | T helper cell |
tlpp | Lipoprotein terminator |
TLR-2 | Toll-like receptor 1 |
TLR-9 | Toll-like receptor 2 |
TMAE | Trimethylaminoethyl |
tmax | Time to reach Cmax |
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Parameter | Tα1-PAS |
---|---|
Cmax (mg/L) | 25.6 ± 4.4 |
tmax (h) | 22.7 ± 1.1 |
AUC0-∞ (h µg/mL) | 1586.7 ± 295.1 |
τ1/2α (h) | 15.7 ± 0.8 |
τ1/2β (h) | 15.9 ± 0.9 |
CL (mL/h/kg) | 2.2 ± 0.4 |
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Binder, U.; Skerra, A. PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology. Int. J. Mol. Sci. 2021, 22, 124. https://doi.org/10.3390/ijms22010124
Binder U, Skerra A. PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology. International Journal of Molecular Sciences. 2021; 22(1):124. https://doi.org/10.3390/ijms22010124
Chicago/Turabian StyleBinder, Uli, and Arne Skerra. 2021. "PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology" International Journal of Molecular Sciences 22, no. 1: 124. https://doi.org/10.3390/ijms22010124
APA StyleBinder, U., & Skerra, A. (2021). PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology. International Journal of Molecular Sciences, 22(1), 124. https://doi.org/10.3390/ijms22010124