Purification of Myristoylated and Nonmyristoylated Neuronal Calcium Sensor-1 Using Single-Step Hydrophobic Interaction Chromatography

2000 ◽  
Vol 20 (1) ◽  
pp. 66-72 ◽  
Author(s):  
Jon R. Fisher ◽  
Yogendra Sharma ◽  
Sheila Iuliano ◽  
Robert A. Piccioti ◽  
Dimitri Krylov ◽  
...  
Keyword(s):  
Hydrophobic Interaction ◽  
Hydrophobic Interaction Chromatography ◽  
Single Step ◽  
Calcium Sensor ◽  
Neuronal Calcium Sensor

Single-Step Separation of Lactate Dehydrogenase Using Thiophilic Chromatography

1998 ◽  
Vol 63 (6) ◽  
pp. 851-856
Author(s):  
Milena Kmínková ◽  
Jiří Kučera
Keyword(s):  
Lactate Dehydrogenase ◽  
Proteolytic Activity ◽  
Cyprinus Carpio ◽  
Hydrophobic Interaction ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Single Step ◽  
Hydrophobic Adsorption ◽  
Negative Adsorption ◽  
Final Purification

Lactate dehydrogenase (EC 1.1.1.27) from carp (Cyprinus carpio) hepatopancreas was purified by the single-step chromatography on thiophilic sorbent. Hydrophobic negative sorption was used as negative adsorption step for final purification. Final specific activity was 8.88 U/mg protein and the yield 84%. The enzyme is not adsorbed during hydrophobic interaction chromatography, but proteolytic activity is adsorbed completely on hydrophobic sorbent. Thus hydrophobic adsorption can be used as a prepurification step.

scholarly journals A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins

Biomolecules ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1025
Author(s):  
Vasiliy I. Vladimirov ◽  
Viktoriia E. Baksheeva ◽  
Irina V. Mikhailova ◽  
Ramis G. Ismailov ◽  
Ekaterina A. Litus ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Hydrophobic Interaction Chromatography ◽  
Bacterial Expression ◽  
Calcium Sensor ◽  
Post Translational Modification ◽  
Growth And Survival ◽  
Neuronal Calcium Sensor ◽  
Novel Approach ◽  
Wide Range ◽  
Sensor Proteins

N-terminal myristoylation is a common co-and post-translational modification of numerous eukaryotic and viral proteins, which affects their interaction with lipids and partner proteins, thereby modulating various cellular processes. Among those are neuronal calcium sensor (NCS) proteins, mediating transduction of calcium signals in a wide range of regulatory cascades, including reception, neurotransmission, neuronal growth and survival. The details of NCSs functioning are of special interest due to their involvement in the progression of ophthalmological and neurodegenerative diseases and their role in cancer. The well-established procedures for preparation of native-like myristoylated forms of recombinant NCSs via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. Here, we report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Ca2+-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.

scholarly journals A new preparative method for isolation of human erythropoietin with hydrophobic interaction chromatography

Blood ◽  
1980 ◽  
Vol 56 (4) ◽  
pp. 620-624 ◽  
Author(s):  
S Lee-Huang
Keyword(s):  
Hydrophobic Interaction ◽  
Hydrophobic Interactions ◽  
Guanidine Hydrochloride ◽  
Phenyl Group ◽  
Hydrophobic Interaction Chromatography ◽  
Preparative Method ◽  
Single Step ◽  
Amino Acid Residues ◽  
Human Erythropoietin ◽  
Pi Interactions

Abstract A new preparative method for isolation of human urinary erythropoietin (Ep) has been developed using hydrophobic interaction chromatography on Phenyl-Sepharose CL4B. Crude urine and urine concentrates from anemic patients were used directly without prior manipulation. In addition to facilitating hydrophobic interactions, Phenyl-Sepharose provided pi-pi interactions between its phenyl group on the gel matrix and the aromatic amino acid residues of Ep, and thus contributed to specific resolution. Over 90% of the urinary contaminants were excluded from the column, and Ep was selectively bound. Its activity was eluted with 20% ethylene glycol in 10 mM NaOH containing 4 M guanidine hydrochloride. This single step offered a mean purification factor of 110 with a recovery of 85%.

scholarly journals A new preparative method for isolation of human erythropoietin with hydrophobic interaction chromatography

Blood ◽  
1980 ◽  
Vol 56 (4) ◽  
pp. 620-624
Author(s):  
S Lee-Huang
Keyword(s):  
Hydrophobic Interaction ◽  
Hydrophobic Interactions ◽  
Guanidine Hydrochloride ◽  
Phenyl Group ◽  
Hydrophobic Interaction Chromatography ◽  
Preparative Method ◽  
Single Step ◽  
Amino Acid Residues ◽  
Human Erythropoietin ◽  
Pi Interactions

A new preparative method for isolation of human urinary erythropoietin (Ep) has been developed using hydrophobic interaction chromatography on Phenyl-Sepharose CL4B. Crude urine and urine concentrates from anemic patients were used directly without prior manipulation. In addition to facilitating hydrophobic interactions, Phenyl-Sepharose provided pi-pi interactions between its phenyl group on the gel matrix and the aromatic amino acid residues of Ep, and thus contributed to specific resolution. Over 90% of the urinary contaminants were excluded from the column, and Ep was selectively bound. Its activity was eluted with 20% ethylene glycol in 10 mM NaOH containing 4 M guanidine hydrochloride. This single step offered a mean purification factor of 110 with a recovery of 85%.

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