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

Abstract

<jats:p>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 Са2+-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.</jats:p>

Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling

“…Expression and purification of recombinant NCS-1 were performed according to a recently proposed procedure, yielding protein product with a high degree of N-terminal myristoylation (~98%) [106]. Fusion protein GRK1 N-C with C-terminal His-tag was expressed in E. coli and purified from bacterial lysate under non-denaturating conditions as described in [26].…”

Zinc Modulation of Neuronal Calcium Sensor Proteins: Three Modes of Interaction with Different Structural Outcomes

“…Plasmids for bacterial expression of recoverin (in pET11d) and NCLD (in pET22b) were constructed in our previous studies [ 47 , 48 ]. GCAP1 and GCAP2 expression vectors were kindly provided by our colleagues from the team of Prof. K.-W. Koch in the University of Oldenburg (Oldenburg, Germany) [ 49 ].…”

“…Molecular cloning of VILIP1 into pET22b plasmids was performed based on total mRNA from bovine brain, using a pair of specific primers: 5′-GGG CAT ATG GGG AAA CAG AAT AGC AAA CTG GC-3′ (direct, NdeI) and 5′-GGG AAG CTT CAT TTC TGA ATG TCG CAC TGC-3′ (reverse, HindIII). Expression and purification of the myristoylated proteins was performed according to previously published procedures [ 47 ]. Myristoylation rate was assessed with analytical HPLC using a reversed-phase column, as described in [ 47 ], and was more than 95%.…”

“…Expression and purification of the myristoylated proteins was performed according to previously published procedures [ 47 ]. Myristoylation rate was assessed with analytical HPLC using a reversed-phase column, as described in [ 47 ], and was more than 95%. All proteins were subjected to a standardized decalcification procedure to obtain the apo-forms [ 36 ].…”

Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin

“…Recombinant myristoylated recoverin and recoverin C39D mutant were obtained as described previously [ 35 , 37 , 41 ]. The myristoylation rate was assessed using analytical high performance liquid chromatography (HPLC) [ 42 ] and was more than 95%. The concentration of recoverin forms was measured spectrophotometrically using a molar extinction coefficient at 280 nm, calculated according to Pace et al [ 43 ].…”