Expression and purification of proteins

CD2.RUBCa and its mutant. The engineered proteins CD2.RUBCa and its mutant CD2.RUBCa.AA were expressed as GST fusion proteins in

Escherichia coli BL21 (DE3) transformed with the plasmid pGEX2T-CD2.RUBCa

or pGEX2T-CD2.RUBCa-AA in LB medium with 100 mg/L of ampicillin and grown at 37 °C. 100 µM of isopropyl-β-D-thiogalactopyranoside (IPTG) was added when the O.D.600 reached 0.6 to induce protein expression for another 3 to

4 hours at 37 °C. The cultures were centrifuged at 7,000 rpm at 4 °C with a Sorvall centrifuge equipped with a SG-3 rotor. The harvested cell pellets were resuspended in lysis buffer consisting of 0.2% sarcosine, 1mM DTT, 1 mM AEBSF in PBS, pH 7.4. The resuspended solution was sonicated 6 times with each time 20 seconds with 80% duty. After centrifugation at 17,000 rpm with a Sorvall centrifuge equipped with a SS-34 rotor at 4 °C, the clarified supernatant was passed through a flow-through affinity column loaded with 4-5 mL slurry GS4B beads (GE Healthcare). After a minimum of 10 bead-volume of washing with PBS, on-column cleavage was performed to remove the GST tag by adding 20 units of thrombin to each column. The elutant containing the target protein was filtered through 0.45 µM filter and injected into Superdex 75 gel filtration column. The eluted proteins were pooled together and further purified by Hitrap SP cation exchange chromatography. The molecular weight of CD2.RUBCa and its mutant were also confirmed by MALDI-TOF-MS. The concentration was determined by using the absorption at 280 nm with an extinction coefficient of 11,700 M-1 cm-1 (61).

RUBCa (Fig. 2.1). The RUBCa was expressed as a GST fusion protein in

Escherichia coli BL21 (DE3) transformed with the plasmid pGEX-2T-RUBCa in

LB medium with 100 mg/L of ampicillin and grown at 37 °C. 100 µM of isopropyl- β-D-thiogalactopyranoside (IPTG) and 50 µM of ZnCl2 were added when the

O.D.600 reached 0.7 to induce protein expression for another 3 to 4 hours. The

proteins were purified following the protocols for GST-fusion protein purification (60) using glutathione sepharose 4B beads (GE Healthcare). The protein was cleaved from its GST tag on beads by taking advantage of the thrombin cleavage site and eluted. The elutants containing RUBCa were further purified using Superdex 75 and Hitrap SP columns (GE Healthcare). The molecular weight of RUBCa was confirmed by MALDI-TOF-MS in the Advanced Biotechnology Core Facilities of Georgia State University. The concentration of RUBCa was measured by its absorption at 280 nm with an extinction coefficient of 19,630 M-1 cm-1, which was calculated according to previously described methods (62).

CaM. Recombinant rat CaM was expressed in Escherichia coli strain BL21(DE3)pLysS transformed with the plasmid pET7-CaM that harbors the synthetic CaM gene (63). pET7-CaM transformed cells were grown in LB medium to obtain unlabeled CaM. 15N-labeled CaM was expressed in SV minimal medium using 0.5 g/L 15NH4Cl (Cambridge Isotope Laboratories, MA, USA) as

the sole nitrogen source. Bacterially-expressed CaM was purified by phenyl- Sepharose (Sigma, MO, USA) chromatography as previously described. The purity of CaM was examined by mass spectrometry or SDS-PAGE (Fig. 2.2). The concentration of CaM was determined by using the ε276 of 3,030 M-1cm-1 (64).

A

B

C

Figure 2.1. The cloning, expression and purification scheme of GST fusion proteins. (A), Target genes are inserted into EcoR I and BamH I digestion sites of pGEX-2T vector which harbors a GST tag that could be removed by thrombin. (B), A typical purification scheme of GST fusion proteins. (C), SDS- PAGE, elution profiles and MS spectrum of the purified protein RUBCa. The SDS-PAGE (left) shows the cleavage of GST fused RUBCa with thrombin for 1 h (lane 1) and 3 h (lane 2), as well as the elutants containing target proteins (Lane 3). The elutants are further purified to homogeneity by gel filtration (Superdex) and cation exchange (SP) chromatography (middle). The molecular weight is finally confirmed by MALDI-TOF-MS (right).

E.Coliculture Harvest cell paste Cell disruption by sonication Centrifugation Supernatant GS4B Affinity column Superdex Gel filtration column

SP cation exchange column On-column cleveage

with thrombin

E.Coliculture Harvest cell paste Cell disruption by sonication Centrifugation Supernatant GS4B Affinity column Superdex Gel filtration column

SP cation exchange column On-column cleveage

Dansyl CaM was prepared according to the method of Johnson et al with slight modifications (65). Briefly, rat CaM was dansylated in the dark by mixing 1 ml protein (1 mM) with a 5-fold molar excess of dansyl chloride (dissolved in 1:1 acetone/ethanol) in 10 mM Mops, 100 mM KCl, 1 mM CaCl2, pH 7.0 for 16 h at

4 °C. The reaction mixture was then extensively dialyzed against 10 mM Tris, 100 mM KCl at pH 7.4 to remove the residual free dansyl chloride. The modification of CaM by dansyl chloride was confirmed by ESI-MS with an increase of +233 in the molecular mass. The bound dye concentration was determined by using the ε 335 of 3980 M-1 cm-1 (65). An average of ~0.8 mol of the

dansyl chromophore was incorporated into per mol of CaM.

Peptides. The peptides Cx43136-158 (Ac-KYGIEEHGKVKMRGGLLRTYIIS-

NH2) and Cx44129-149 (Ac-VRDDRGKVRIAGALLRTYVFN-NH2) were synthesized

by Sigma-Genosys (Sigma, USA) and purified by preparative reversed-phase HPLC with purity over 95%. A randomized control peptide (Ac- LGGEYLVTMESKIHIKGKRIGYR-NH2), with the same composition of amino

acids as Cx43136-158 but arranged in a different order, was similarly synthesized.

The molecular weight of the synthetic peptide was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. To mimic its protein environment and eliminate extra charges, the designed peptide was blocked at its N-terminus with an acetyl group and at its C-terminus with an amide group.

A

B

Figure 2.2. SDS-PAGE (A) and MS (B) spectrum of purified CaM. Upon binding to Ca2+, the CaM undergoes conformational changes by switching from a globular shape at its apo-form to an elongated dumbbell shape, and therefore, moves faster in the electric field. The measured molecular weight (16706.98 Da) matches well with the calculated theoretical molecular mass (16707 Da). 1999.0 9599.4 17199.8 24800.2 32400.6 40001.0 Mass (m/z) 0 8117.8 0 10 20 30 40 50 60 70 80 90 100 % I n t e n s it y Voyager Spec #1[BP = 16707.3, 8118] F2CaM_1:10CHCA_linear positive_070605 16706.98 8353.81 16914.04 33411.41