Journal of Biotechnology 58 (1997) 187 195

Rapid SDS-GEL capillary electrophoresis for the analysis of recombinant NADP + -dependent formate dehydrogenase during expression in Escherichia coli cells and its purication
V. Klyushnichenko *, V. Tishkov, M.-R. Kula
Institute of Enzyme Technology, Heinrich -Heine Uni6ersity Du sseldorf, D -52426 Ju lich, Germany Received 23 June 1997; received in revised form 16 September 1997; accepted 22 September 1997

Abstract The level of expression in Escherichia coli cells and different steps of purication of the recombinant NADP + -dependent formate dehydrogenase (EC 1.2.1.2, FDH) from bacterium Pseudomonas sp.101 was analyzed by rapid SDS-Gel capillary electrophoresis (SDS-Gel CE) and compared with SDS polyacrylamide gel electrophoresis (SDS PAGE). First standard proteins were separated in the short capillary and the calibration curve generated, then fractions taken during the fermentation and purication process were analyzed. The main advantages of SDS-Gel CE are short analysis time, high sensitivity, the possibility to quantify proteins at different ultraviolet wavelength, and small injection volumes. The data for each step of the fermentation process and during the purication were controlled by spectrophotometric analysis of enzyme activity and protein concentration as well as standard SDS PAGE. The molecular mass of the puried FDH was determined as 44 078 Da by matrix-assisted laser desorption/ ionisation time of ight mass spectrometry 1997 Elsevier Science B.V. Keywords: Capillary electrophoresis; SDS-Gel capillary electrophoresis; Proteins; Recombinant formate dehydrogenase; Rapid analysis; MALDI-TOF-MS, matrix-assisted laser desorption/ionisation time of ight mass spectrometry

1. Introduction One of the main parameters to estimate the level of protein expression during the cell cultiva* Corresponding author. Tel.: + 49 2461 612938; + 49 2461 612947; fax: + 49 2461 612490; e-mail: klyushni@ibt022. ibt.kfa-juelich.de

tion and following purication is the distribution of protein molecular mass (MM), which can be gained by different chromatographic, electrophoretic and spectrometric techniques. The analysis by SDS-Gel capillary electrophoresis (SDS-Gel CE) can be fast, sensitive for small differences in the MM of reduced and aggregated proteins in a wide MM range (Tsuji, 1994), and

0168-1656/97/$17.00 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 8 - 1 6 5 6 ( 9 7 ) 0 0 1 4 9 - 1

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besides requires very small sample and buffer volumes (Kinghorn et al., 1995). Among different application of SDS-Gel CE the separation of complex protein mixtures of recombinant and/or native proteins (Takagi and Karim, 1995) from biological origins (Nakatani et al., 1993) are dominating. NAD + -dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methanol-utilizing microorganisms catalyses the oxidation of formate to carbon dioxide with the corresponding reduction NAD + to NADH. This enzyme provides the best NADH regeneration systems used in the processes of enzymatic synthesis of chiral and physiologically active compounds (Hummel and Kula, 1989a). Large scale production of NAD + -specic FDH from the yeast Candida boidinii was developed a few years ago (Wuester-Botz et al., 1994) and this enzyme was used for the industrial production of tert-leucine and can be applied together with many other dehydrogenases (Kragl et al., 1996). Normally, in nature in coupled enzymatic systems NAD(H) is involved in biodegradation processes and NADP(H) in biosynthesis. Unfortunately a NADP + -specic FDH not requiring complex metal clusters has not been found in living cells yet. A NADP + -dependent enzyme has been generated, however, by the method of site-directed mutagenesis of the FDH gen from the bacterium Pseudomonas sp.101 (Tishkov et al., 1993) and rst model experiments of coupled enzyme catalysed synthesis with NADPH regeneration have been reported (Seelbach et al., 1996). Overexpression and large scale productions of this mutant FDH are necessary for industrial realization of such processes. Here we describe rapid SDS-Gel CE as a method to analyze FDH expression in E. coli cells during cultivation and in further enzyme purication steps.

by Tishkov et al. (1996). The cells were cultivated in 1.3 l of 2YT medium (per liter16 g of bactotrypton, 10 g of yeast extract (both from Difco, Detroit) and 5 g of NaCl) in 5 l akes at 37C and 150 rpm. At certain time intervals probes were taken and analyzed as described below. At the end of the cultivation cells were separated at 5000 rpm by centrifugation in Sorvall RC-5B centrifuge (Du Pont, Bad Homburg) and stored frozen at 20C if not used immediately.

2.2. Probe preparation

In 2 ml Eppendorf tube, 1.5 ml of culture was sedimented for 30 s at 14 000 rpm using a Eppendorf 5415C Centrifuge (Eppendorf-Netheler-Hinz GmbH, Hamburg). The biomass was resuspended in 0.75 ml of 0.1 M potassium phosphate, 0.01 M EDTA (pH 7.0) buffer and desintegrated by wet milling in a Retsch MM2 desintegrator (Kurt Retsch GmbH, Haan) with 1 g of glass beads of 0.20.3 mm diameter for 10 min (Hummel and Kula, 1989b). Then the biomass was centrifuged 1 min at 14 000 rpm and the supernatant used for enzyme activity measurement and preparation of probes for the capillary electrophoresis. A total volume of 40 v l of the crude extract was mixed with 10 v l of 15% SDS, 10 v l 1 M dithiotreitol (DTT) and 40 v l H2O. The mixture was heated for 5 min at 95C, then put into ice for 30 s to cool to room temperature and applied to the CE. Enzyme activity was measured spectrophotometrically at the 340 nm in 0.1 M K-phosphate buffer (pH 7.0), at 30C. The concentrations of NADP + and sodium formate in the reaction mixture were 1.5 mM and 0.3 M, respectively (Tishkov et al., 1993).

2.3. Purication of FDH

2. Materials and methods Cells (30 g wet wt.) were resuspended in 300 ml of 50 mM Tris-HCl, 50 mM glucose, 15 mM EDTA, (pH 8.0) buffer and frozen at 20C overnight. After thawing egg white lysozyme was added to a nal concentration of 50 mg l 1 and the solution kept for 1 h at 37C in a shaker. Cell debris was removed by centrifugation at 12 000

2.1. Protein expression

For the expression of NADP + -dependent FDH we used the same system as for the expression of the wild-type NAD-specic enzyme as described

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rpm in Sorvall RC-5B centrifuge (Du Pont, Bad Homburg). Solid ammonium sulfate was added with stirring to the supernatant to give 30% of saturation and the suspension kept for 3 h at room temperature. The precipitate was removed by centrifugation at 12 000 rpm and more ammonium sulfate was added to reach 75% of saturation and the mixture left overnight at + 4C. The precipitate (FDH) was collected by centrifugation, washed with a solution of ammonium sulfate (75% of saturation) in 0.1 M K-phosphate, 15 mM EDTA buffer, pH 7.0 (buffer A), redissolved in buffer A and applied on a 50 ml column packed with phenyl-sepharose fast ow (Pharma-

Fig. 2. SDS PAGE separation of fractions during the cell cultivation and nal puried FDH: (a): marker proteins; (b): samples after 13.5 h; (c): 14.5 h; (d): 15.5 h; (e): 16.5 h; (f): 17.5 h; (g): of cultivation, puried FDH.

cia AB, Uppsala) and equilibrated with ammonium sulfate (30% of saturation) in buffer A. The enzyme was eluted from the column by a step change to a 4% of ammonium sulfate saturation in buffer A. Final purication was done on a 1.5 l column with Superdex G-200 (Pharmacia AB, Uppsala) equilibrated with buffer A. Elution was performed at a ow rate of 1 ml min 1.

2.4. Capillary electrophoresis

All CE measurement were performed with a P/ACE 2100 device for capillary electrophoresis (Beckman, Fullerton, CA). Prior to each analytical run a bare silica capillary with modication according to Hjerten (1985) (27 cm length, 7 cm effective length) 100 v m I.D. 375 mm O.D.) was rinsed with 1.0 M HCl water for 2 min followed by a lling with the SDS polymer solution (eCAP SDS 14-200, Beckman) for 2 min. The column temperature was maintained at 20C by circulating a coolant to minimize band diffusion and ensure effective size separation. An electrophoretic run was conducted at 300 V cm 1 (8.1 kV for the capillary) using the SDS polymer solution (Beckman). The molecular mass protein standard were injected for 20 sec (5 kV) using the electrokinetic mode into the SDS polymer-lled capillary column as described in Klyushnichenko and Kula (1997a).

Fig. 1. (a): Separation of standard proteins by SDS-Gel CE, 27 cm coated capillary, 8.7 kV, 20 s electrokinetic injection at 5 kV. (b): Calibration curve of the separation of the standard proteins: 1, lysozyme (14 kDa); 2, trypsin-inhibitor (20 kDa); 3, triose phosphate isomerase (26.6 kDa); 4, aldolase (39 kDa); 5, glutamate dehydrogenase (55.5 kDa); 6, fructose-6-phosphate kinase (85.2 kDa); 7, i -galactosidase (116.3 kDa), 8, IgG (150 kDa).

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Fig. 3. Analysis of expression of FDH at different times of cell cultivation: (a): 13.5 h; (b): 14.5 h; (c): 15.5 h; (d): 16.5 h; (e): 17.5 h. Panel A monitored at 214 nm, panel B monitored at 280 nm. Conditions: 8.1 kV, 40 50 v A, 1 kV.

Model proteins were dissolved in the buffer for SDS Gel CE: 0.1 M Tris-HCl, 1% SDS (pH 6.6). For calibration and analysis the following proteins were used: lysozyme (14.0 kDa), trypsininhibitor (20 kDa), triose phosphate isomerase (26.6 kDa), aldolase (39 kDa), glutamate dehydrogenase (55.5 kDa), fructose-6-phosphate kinase (85.2 kDa), i -galactosidase (116.3 kDa), (Boehringer Mannheim, Mannheim) and IgG (150 kDa) (Institute of Enzyme Technology, University of Dusseldorf). Before analysis all buffers and samples were degassed and ltered through 0.22 0.45 v m lters.

2.5. SDS polyacrylamide gel electrophoresis (PAGE)

This was performed in 10% polyacrylamide (0.8% bisacrylamide) according to Laemmli (1970).

2.6. Matrix -assisted laser desorption /ionisation time of ight mass spectrometry (MALDI -TOF -MS)
The measurements were performed with an instrument constructed in the Institute of Laser

V. Klyushnichenko et al. / Journal of Biotechnology 58 (1997) 187195 Table 1 Analysis of protein concentration, FDH activity and specic activity at different times of the cell cultivation Time (h) FDHa conc. in probe (mg ml1) 0.257 0.543 0.567 0.556 0.561 FDH activity in probe (U ml-1) 0.644 1.355 1.400 1.376 1.389 Specic activity (U mg1) 2.51 2.49 2.47 2.48 2.48 Cell absorbance A560 (optical units) 13.0 13.9 14.2 14.7 14.5

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13.5 14.5 15.5 16.5 17.5

a

Concentration of FDH in probe was determined from CE experiments at 214 nm (Fig. 2) using a calibration curve with BSA as a standard (data not shown). The specic activity of FDH was calculated as the ratio of enzyme activity to FDH concentration in the probe (column 3 and 2, respectively).

Medicine (Du sseldorf University) in the linear time-off-ight mode with the laser beam wavelength u = 337.1 nm (Spengler et al., 1990). The samples were prepared in 2,5-dihydroxybenzoic acid with 10% of 2-hydroxy-5-methoxybenzoic acid as described by Karas et al. (1993). All chemicals used were at least analytical grade if not stated separately.

3. Results and discussion Usually in the commercial instruments the capillary has a long (20 50 cm) and a short (2 10 cm) part connected by a window for the detection. The separation is usually performed in the long part of the capillary. But in this case the time of analysis is about 15 50 min. The separation in the short part (7 cm) of the capillary was investigated earlier for rapid separation of proteins (4 5 min) with high reproducibility during hybridoma cell cultivation (Klyushnichenko and Kula, 1997a,b). The separation of the standard proteins for the MM estimation was performed before measurements (Fig. 1a). The protein peaks with different MM are separated up to baseline with high resolution. The separation of proteins occurs in the interval between 3 and 4.5 min. The peaks before 3 min arise from baseline noise and low molecular mass impurities. The calibration curve was generated as a function of l g MM versus the elution time. It should be noted, that the calibration curve is semi-linear in the MM range 14 150

kDa. Due to the different nature of the proteins as charge, hydrophobicity and structure (glycoproteins) they differ in the interaction with SDS and some times migrate unexpectedly in the SDSgel solution (Werner et al., 1993). The fractions during the cultivation of E. coli harboring the FDH gene were taken and analyzed in parallel by PAGE (Fig. 2) and SDS-Gel CE (Fig. 3). The probes were analysed in the CE at two wavelengths in the UV region at 214 and 280 nm. At the time of analysis the cells entered the stationary phase of growth and the concentration of FDH is increasing rapidly between 13 and 14.5 h of the cell cultivation. After 14.5 h the FDH content became constant and the cultivation was stopped. Data in Table 1 show that there are no big differences in protein content between the time of cultivation 14.517.5 h. The recombinant mutant FDH is stable in the E. coli cells. As the protein content remains at plateau values, a proteolytic degradation of the proteins is not observed. The protein folding in the cells occurs fast, as evidenced by the constant relation of the FDH specic activity at the different times (Table 1). Using SDS PAGE separating proteins with big differences in the concentration the problem of quantication of main protein and impurities arise, which is illustrated in Fig. 2. Even if the content of the main protein is about 5060% the remaining 4050% of the other proteins are distributed over different MM (Fig. 2, lanes bf). If one wants to see a main protein and impurities with close MM, as products of its degradation, or other impurities, different amount should be ap-

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Fig. 4. Analysis of FDH samples at different stages of purication. Conditions: 8.1 kV, 40 50 v A, 1 kV (A), 5 kV (B). Panel A seperation monitored at 214 nm, panel B seperation monitored at 280 nm. (a): Cell extract; (b): ammonium sulfate, 30% saturation; (c): ammonium sulfate, 75% saturation; (d): after purication on phenyl-sepharose; (e): after gel ltration through Superdex G-200; (f): buffer alone (only A).

plied. The registration and quantication is difcult even employing a laser gel scanner. In our case we have overloaded the SDS PAGE in order to show the impurities. Compared to the SDS PAGE separation the concentration range of the SDS-Gel CE and sensitivity is much higher. Otherwise there is a full correspondence of the main peak and the impurities between the SDS PAGE and the SDS-Gel CE separations (Figs. 2 4). The detection at 214 nm is unique for proteins and it is possible to compare all steps of the fermentation process registered by both methods (Fig. 2, lanes b f; Fig. 3b, graphs ae). Even with detection at 280 nm and overloading the capillary resulting in an asymmetric peak of FDH, it is possible to quantify main

impurities (3.13.3 min), and their strong reduction in the process of purication (Fig. 4b) is evident. For the analysis of the FDH after different steps of purication with detection at 280 nm a higher voltage was applied during the injection because of the lower (compare to 214 nm) sensitivity. At 214 nm all impurities are registrated and corresponded with the SDS PAGE. We have overloaded the capillary with the puried FDH (Fig. 4a, graph e) at 214 nm in order to show the protein purity and the presence of impurities, if any. The little peak at 3.85 min corresponds with the thin high MM band (about 90 kD) visible on SDS PAGE (Fig. 2, lane g), the early peaks B 3 min correspond to the injection and buffer impurities.

Table 2 Results of FDH purication Total protein (mg) 2380 2140 1570 1060 856 2510 2140 2.37 2.50 2880 1.83 3100 3100 1.30 1.45 1.00 1.12 1.41 1.82 1.92 Total a activity (U) Specic activity (U mg1) Purication (-fold) Purity by SDS-Gel CE (%) 56 67 77 94 98 Yield (%) 100 100 93 81 69

Purication step

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Cell-free extract Ammonium sulphate (30% of satn.) Ammonium sulphate (75% of satn.) Phenyl sepharose Superdex G-200

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Acknowledgements V. Klyushnichenko and V. Tishkov gratefully acknowledge the Alexander von Humboldt Foundation for nancial support of this work. We thank Prof. F. Hillenkamp and Dr K. Strupat (Institute of Medical Physics, University of Muenster) for advice and Dipl. Ing. A. Rodenbrock for expert help with the MALDI-TOF-MS experiments.

References
Fig. 5. MALDI-TOF-MS of pured FDH. The relative intensity is plotted as molecular mass (MM) per unit charge (z ). Hjerten, S., 1985. High-performance electrophoresis elimination of lectroosmosis and solute adsorption. J. Chromatogr. 347, 191 198. Hummel, H., Kula, M.-R., 1989a. Dehydrogenases for the synthesis of chiral compounds. Eur. J. Biochem. 184, 1 13. Hummel, W., Kula, M.-R., 1989b. Simple method for smallscale disruption of bacteria and yeast. J. Microbiol. Methods 9, 201 209. Karas, M., Ehring, H., Nordhoff, E., Stahl, B., Strupat, K., Hillenkamp, F., Grehl, M., Krebs, B., 1993. Matrix-assisted laser desorption/ionization mass spectrometry with additives to 2,5-dihydroxybenzoic acid. Org. Mass Spectrom. 28, 1476 1481. Kinghorn, N.M., Norris, C.S., Paterson, G.R., Otter, D.E., 1995. Comparison of capillary electrophoresis with traditional methods to analyse bovine whey proteins. J. Chromatogr. 700, 111 123. Klyushnichenko, V., Kula, M.-R., 1997a. Rapid SDS Gel Capillary Electrophoretic analysis of proteins. J. Capillary Electrophoresis 2, 61 64. Klyushnichenko, V., Kula, M.-R., 1997b. Sodium dodecyl sulfate polymer capillary electrophoresis for the analysis of cell culture proteins. Electrophoresis 18, 2019 2023. Kragl, U., Kruse, W., Hummel, W., Wandrey, C., 1996. Enzyme engineering aspects of biocatalysis: cofactor regeneration as example. Biotechnol. Bioeng. 52, 309 319. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685. Nakatani, M., Shibukawa, A., Nakagawa, T., 1993. Sodium dodecyl sulfate-polyacrylamide solution-lled capillary electrophoresis of proteins using stable linear polyacrylamide-coated capillary. Biol. Pharm. Bull. 16, 1185 1188. Seelbach, K., Riebel, B., Hummel, W., Kula, M.-R., Tishkov, V.I., Egorov, A.M., Wandrey, C., Kragl, U., 1996. A novel, efcient regeneration method of NADPH by using new formate dehydrogenase. Tetrahedron Lett. 37, 1377 1380.

A summary of the fermentation and purication process is shown in Table 2. The initial cell extract contained usually about 56% of FDH (Fig. 4, graph a). After addition of 30% of ammonium sulfate, some protein impurities are precipitated and removed (Fig. 4, graph b). At the 75% of saturation with ammonium sulfate the precipitate contained 77% FDH (Fig. 4, graph c). After redisolving the protein mixture was applied to the hydrophobic interaction and size-exclusion chromatography. The nal purity of the protein was better than 98%. The puried protein was analyzed by MALDITOF-MS (Fig. 5). The MM of the FDH was found to be 44 078 Da, which corresponds to the expected value, based on DNA sequence information. In the scan some additional peaks are visible (Fig. 5) corresponding to the double, and triple charged molecules and to the single charged dimer of FDH, respectively. The error in the single measurement was about 0.5%, and the average value of ve measurements is quoted.

4. Conclusion Rapid SDS-Gel CE was successfully applied to follow the expression of a recombinant protein in E. coli and to quantify protein purication. Due to the high sensitivity and speed SDS-Gel CE is a powerful tool in the process development.

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