Protein isolation

For convenient preparation of proteins, a heterologous system was established by Zhao et al. (2002), using the expression vector pET-30a (Novagen, Schwalbach) with intrinsic kanamycin-resistance and D-galactose-inducible promoter. Additionally, His6-tagged derivatives were constructed to allow for easy purification of the gene

products.

2.2.2.1 Protein expression in Escherichia coli

The pET-based plasmids were used to transform E. coli BL21(DE3). Cells were grown

in Luria-Bertani (LB) medium containing kanamycin (see Table 2-2 for antibiotic concentrations) at 37 °C for genes from M. laminosus or at 20 °C for genes from

Anabaena sp. PCC 7120. For expression of untagged proteins, which is available with

the Duet vectors (Novagen, Germany), kanamycin has to be substituted by other antibiotics in different final concentrations (see Table 2-2).

Once the cultures have reached the exponential growth phase (after 4-5 hr, A600 =

0.5–0.7), expression of the foreign gene is induced with IPTG (1mM). Cells were collected by centrifugation (7000 xg, 15min) 5 hr after induction (for genes from M.

laminosus) or after 6 hr (for genes from Anabaena sp.), then washed twice with

doubly distilled water and stored at -20 °C until use.

2.2.2.2 Protein purification

Cell pellets were resuspended in ice-cold start buffer and disrupted by sonication (30 min at 150 W, duty cycle 40%). The suspension was centrifuged at 12,000 xg for 30 min at 4 °C. The crude protein-enriched supernatant was stored at 4 °C until use.

Ni2+_{-affinity chromatography}

Subsequently, the supernatant was purified via Ni2+_{-affinity chromatography on}

chelating Sepharose (Amersham Biosciences). The His6-tag coordinates the bivalent

cation and is adsorbed to the column, while the major fraction of the bacterial proteins has generally no or little affinity for the column material. 30 ml of the supernatant were applied to a Ni2+_{- chelating column (60 x 20 mm) equilibrated with start buffer (see Tab.}

2-1 for all solutions and buffers), and then were washed with start buffer and elution buffer. The following washing steps increase in stringency to enhance the purification

process. To finally elute the product fraction, high imidazole concentration (0.5-1M) is applied, which is a strong chelator of Ni2+ ions, thus displacing the histidines and releasing them from the stationary phase.

After collection, the protein fractions were dialyzed twice against the initial buffer and kept at -20 °C until use.

For regeneration of the column, EDTA buffer is used to strip off Ni2+ _{and protein}

remainders. After excessive rinsing with water, Ni2+_{-solution is applied to recharge the}

column. Consecutive equilibration with starting buffer prepares the column for application of another protein sample. In case the EDTA buffer is not sufficient to clean the column (when a “dirt veil” is still visible after the rinsing step), a washing step with 0.5 M NaOH can be included for improved cleaning. If not in use, the column is stored in 20% ethanol.

Fast Performance Liquid Chromatography

Samples (6 ml) in Tris buffer (1 M, pH 6.0) were concentrated to an overall volume of 1 ml using centrifugal filter devices (Amicon Ultra-15, Millipore, USA). They were then applied to the gel column, Superdex 200-16/60 preparative grade (Amersham Biosciences, flow rate 0.75 ml/min) equilibrated with Tris buffer (1 M, pH 6.0). The eluant was monitored with a diode array detector (Tidas, Diode-Array-Photometer, J&M, Germany; 280–800 nm, measurement interval 5 s). Fractions were collected and analyzed by SDS-PAGE according to Laemmli (1970) using 15% stacking and 5% separating gels (Boehm, 2006).

2.2.2.3 Protein analysis

After dialysis, the solutions are retrieved, transferred to Falcon tubes, and their protein contents were evaluated employing two methods:

Sodium dodecyl sulphate polyacrylamide (SDS-PAGE) gels

50 μl of the protein solution are mixed with 50 μl SDS-PAGE loading buffer and boiled for 5 minutes. 10-20 μl of the sample are applied to a two step SDS-PAGE gel (see table 2-1) and run for 30 min of 50 V, followed by 75 min of 150 V. The gel is developed by overnight incubation in staining solution, and subsequently excess dyes removed by destaining solution, until the desired degree of contrast is achieved.

Chromophorylated protein species are identified by Zn2+_{-induced fluorescence prior to}

Coomassie staining: after soaking the gels in 1 M Zn2+ acetate solution for 15 min, UV-light (360 nm) induced fluorescence can be observed (Berkelman and Lagarias, 1986).

Protein assay

Protein concentrations were determined by protein assay kits from Fluka advance (Seelze, Germany).10 μl of the protein solution are mixed with 300 μl FLUKA solution and 690 μl distilled water, and incubated for several minutes. Absorption of the protein sample is measured at 590 nm and the concentration in mg/ml deduced by comparison to an albumin standard. For protein solutions in the expected concentration range of 5-10 mg/ml, as in this case, a 1:10 dilution with distilled water proved to give convenient absorption values. Comparing this value with the corresponding lanes on the SDS-PAGE and taking into account the contamination with co-purified E. coli proteins, a rough estimate of the concentration of the products

is possible. Since the molar masses for the proteins are known, the concentration in mg/ml can be converted to μmol/ml. 1 L expression medium usually yields 10 ml protein solution (5-10 mg/ml in concentration as determined by the FLUKA Protein assay) which are equivalent to 4-6 μmol protein.

2.2.2.4 Concentration of proteins

In case the volume of a protein solution needs to be reduced to increase its relative protein concentration, it is transferred to a centrifuge filter (Biomax 10K 15 ml, Millipore) and centrifuged (4 °C, 7 000 xg) until the desired volume reduction is achieved.

2.2.2.5 Protein storage

The protein solutions are stable at room temperature down to 4°C for approximately 2 days. Long-term storage is possible at -20 °C but accompanied by loss of specific enzyme activity.