Comparison of Soluble Activity of PST Constructs¹.

1<p>Experiments were performed in triplicate (<i>n</i> = 3) with mean values and standard error reported.</p

Stability and thermolability of PST_Nm, PST_Ec, and PST_Mh.

<p>The specific activities of the three bacterial PSTs were monitored during a two-hour time course at three different reaction temperatures (25°C, 30°C, and 37°C). Both PST_Nm and PST_Ec demonstrated optimal temperature at 37°C in constrast to PST_Mh, which demonstrated lower activity at this higher temperature compared to other incubation temperatures. After 10 min incubation there is no longer an advantage for the activity at a higher temperature for these two enzymes. . Enzymes from three separate purification batches were tested in duplicate (<i>n</i> = 6).</p

Biochemical Properties of Bacterial Polysialyltransferases¹.

1<p>Experiments were performed in triplicate (<i>n</i> = 3) with mean values and standard error reported.</p>2<p>Experiments were performed with enzymes from three separate purification batches in duplicate (n = 6) with mean values and standard error reported.</p

Expression and solubility of PST_Mh constructs.

<p>The PST constructs were expressed in <i>E. coli</i> AD202 followed by cell disruption and centrifugation. The soluble (Sup) and insoluble (Pel) fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Bands corresponding to the predicted sizes of the protein variants were observed (indicated with an asterisk). The increased intensity of the bands of the MalE-fusions indicated increased expression compared to the non-fusion protein. Furthermore, the truncated protein (MalE-Δ20PST_Mh) variant demonstrated the best solubility.</p

Polysialylation of cell-surface proteins on live PC12 cells.

<p>PC12 cells were incubated with the three PSTs in the absence and presence of 5 mM CMP-Neu5Ac for 1 hour at 37°C. Cell-surface PSA was detected using PSA-NCAM immunocytochemistry and nuclei were detected with Hoechst 33258 and confocal microscopy imaging. Following treatment of PC12 cells with all three PSTs in the presence of CMP-Neu5Ac specific cell-surface PSA labeling was detected (as indicated in the panels denoted “+CMP-Neu5Ac). Control reactions were also performed with the CMP-Neu5Ac absent; in these reactions PSA labeling was not detected. Quantitation of cellular polysialylation was performed using ImageJ (v 10.2) on the immunocytochemical images from two independent experiments by measuring the fluorescent intensity of 15 randomly selected cells per image. Images were pre-processed by performing background subtraction (50 pixel rolling ball radius) prior to measuring pixel intensity. Relative fluorescence values were calculated by dividing the PSA intensity (green fluorescence) by DAPI intensity (blue fluorescence). Data were normalized to average+CMP fluorescence for each experiment (n = 2).</p

SDS-PAGE Analysis of PST Reactions on diSA-fetuin.

<p>A.The three PSTs were incubated with the acceptor molecule disialyl-fetuin using standard reaction conditions (lanes denoted “A”) as well as a reaction condition to improve the formation of the product (13 µg enzyme, lanes denoted “B”) for 1 or 2 h. A negative control reaction (containing no enzyme) was included (“-C”). The small band at ∼75 kDa indicates the PST added to the reaction. B. Following SDS-PAGE analysis, the proteins were transferred to a membrane and probed with α-PSA antibodies. All reactions were positive for PSA except the negative control lane containing unmodified disialyl-fetuin.</p

Analysis of PSA Chain Length by Capillary Electrophoresis.

<p>Capillary electrophoresis was used to analyze the reactions of GD3-FCHASE and CMP-Neu5Ac catalyzed by the three bacterial PSTs. Shown here are the reactions stopped at approximately 15% conversion. The large peak at ∼9.4 min represents the starting material and subsequent peaks are the products with additional Neu5Ac residues added. Acceptor plus 2 and 10 Neu5Ac products are indicated with arrows. The chain length of the products generated by PST_Nm (18 Neu5Ac) and PST_Mh (16 Neu5Ac) were significantly longer than that of PST_Ec (7 Neu5Ac). The electropherograms of the reactions have been overlaid on the same axis to aid in the comparison.</p