The PCR fragment was purified, cut with NcoI and EcoRI and cloned into the
pKB3 plasmid resulting in pKB3CalB-C311A-His. The sequences of both mutants
...
Supporting Information © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006
Supporting Information for
Site-Specific Immobilization of Genetically Engineered Variants of Candida antarctica Lipase B Kerstin Blank,* Julia Morfill, and Hermann E. Gaub Construction of plasmids for the expression of the CalB variants The plasmids for the periplasmic expression of the different CalB variants are based on the pAK series.[1] Starting point for this study was the plasmid pKB3CalB-His,[2] which contains the calB gene cloned after a lac promotor, the strong RBS T7G10 and a pelB signal peptide for periplasmic expression. Based on this plasmid two different mutants of CalB possessing a free cysteine were generated. For CalB-HisGGC the His tag has been replaced by a tag of six histidines followed by two glycines and a cysteine using the oligonucleotides HisGGC_forw (5’-Pho-AATTCCACCATCATCACCATCATGGCGGATGCTAAT-GAA-3’) and HisGGC_rev
(5’-Pho-AGCTTTCATTAGCATCCGCCATGATGGTGATGATGGTGG-3’).
These oligonucleotides were hybridized and inserted into the plasmid between the EcoRI and HindIII sites resulting in the plasmid pKB3CalB-HisGGC. For CalB-C311A-His the mutation has been introduced by PCR using the following primers (The restriction sites are underlined, the sequence of the calB gene is written in italics and the C311A-mutation in bold.): The forward primer CalB_forw (5’-CATGCCATGGCGGACTACAAAGATCTACCTTCCGGTT CGGACC-3’) contains the sequence of a short FLAG tag[3] and a NcoI site as in the original pAK400 plasmid. The reverse primer CalB-C311A_rev (5’-CCGGAATTCGGGGGTGACGAT GCCGGAGGCGGTCCTTTTGCCTACTG-3’) introduces the mutation and an EcoRI site for cloning. The PCR fragment was purified, cut with NcoI and EcoRI and cloned into the pKB3 plasmid resulting in pKB3CalB-C311A-His. The sequences of both mutants have been verified by sequencing. S1
Expression of CalB variants The plasmids encoding the 3 different variants of CalB (CalB-His, CalB-HisGGC, CalBC311A-His) were transformed in the E. coli K12 strain TB1 (New England Biolabs, Frankfurt, Germany). Small-scale expressions for Western blot analysis were performed at 25 °C using 50 mL of SB medium (20 g l-1 tryptone, 10 g l-1 yeast extract, 5 g l-1 NaCl, 50 m M K2HPO4) containing 30 µg mL-1 chloramphenicol. The cultures for the purification of the lipase variants were grown in 200 mL of SB medium at 25 °C. All cultures were inoculated from a 20 mL preculture to OD600=0.1. Expression was induced with 1 m M Isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 between 1.0 and 1.5. The cells were harvested 3 h after induction by centrifugation at 5000 g and 4° C for 10 min. Western blot analysis The received cell pellets of the small-scale cultures were used for analysis of the expression level of the different lipase variants. Western blots have been performed using antiFLAG antibody M1 (Sigma, Taufkirchen, Germany) and anti-His antibody Penta-His (Qiagen, Hilden, Germany) as described previously.[2] Shortly, the cells were resuspended in PBS buffer and normalized to their end OD600 using 2.5 mL buffer per 1 unit OD600. Whole cell extracts were prepared by French Press lysis. After centrifugation at 16000 g and 4 °C for 60 min, the supernatants containing the soluble proteins (S) were transferred to a new vessel and the pellet (P), representing the fraction of insoluble proteins, was resuspended in the original volume of PBS. SDS-PAGE of both the soluble and pellet fractions was carried out under reducing conditions according to standard protocols using 15 % polyacrylamide gels. The proteins were transferred to a PVDF membrane (Millipore, Eschborn, Germany). For the detection either the anti-FLAG antibody M1 or the anti-His antibody Penta-His were used. Whereas the anti-His antibody was labeled directly with AlexaFluor 647, a secondary rabbit anti-mouse antibody with an AlexaFluor 647 label (Invitrogen, Karlsruhe, Germany) was necessary for the detection of the anti-FLAG antibody. Fluorescence images were taken using a LS300 scanner (Tecan, Crailsheim, Germany). The resulting images are shown in figure S1. Both blots are almost identical and show one strong band of the same size indicating that the three different variants are full-length protein and that no degradation occurs. Comparing the amount of soluble protein for the different variants, the amount of soluble protein is reduced for the variants containing a free cysteine (CalB-HisGGC and CalB-C311A-His).
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Figure S1. Western blots showing the expression levels of the CalB variants E. coli cells were normalized to their end OD600 and lysed. Soluble proteins (S) were separated from the insoluble fraction (P) by centrifugation. The insoluble fraction was resuspended in the original volume. The same volume of soluble and insoluble protein was loaded onto the gel. The CalB variants contain a N-terminal FLAG and a C-terminal His tag. Therefore both antibodies were used to detect the variants. Both blots show a band of the same size. For all three variants CalB-His (wt), CalB-HisGGC (GGC) and CalB-C311A-His (C311A) a certain amount of soluble protein can be detected. In all cases the amount of soluble protein is less compared to the amount of CalB in the insoluble fraction. Comparing the variants with a free cysteine (GGC and C311A) with the original CalB-His (wt), it can be seen that the amount of soluble protein is reduced for these variants. Purification using Immobilized-metal ion affinity chromatography (IMAC) Periplasmic extracts have been prepared according to a protocol included in the manual for the Ni2+-NTA columns (Qiagen, Hilden, Germany). The extracts were dialyzed against loading buffer (50 m M sodium phosphate pH 8.0, 300 m M NaCl, 10 m M imidazole) and loaded onto Ni2+-NTA columns (Qiagen) equilibrated with loading buffer. Columns were washed with 20 column volumes of loading buffer, followed by 10 column volumes of loading buffer containing 1 m M Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Perbio Science, Bonn, Germany) and 5 column volumes of a washing buffer (50 m M sodium phosphate pH 8.0, 300 m M NaCl, 30 m M imidazole). Elution was achieved by adding 5 column volumes of elution buffer (50 m M sodium phosphate pH 8.0, 300 m M NaCl, 200 m M imidazole). The eluted fractions were dialyzed against coupling buffer (50 m M sodium phosphate pH 7.2, 50 m M NaCl, 10 m M EDTA) and concentrated using Centricon YM-10 (Millipore). The actual concentration of the purified lipases was determined by measuring the absorbance at 280 nm. The extinction coefficients of the different CalB variants have been calculated using the program Vector NTI (Invitrogen). Based on these concentrations the yield was calculated for all three preparations: For CalB-His the yield was 2.2 mg per liter culture, for CalB-HisGGC 0.3 mg L-1 and for CalB-C311A-His 0.23 mg L- 1. The purified lipase variants were adjusted to a final
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concentration of 0.6 mg mL-1 and stored in aliquots at -80 °C. Samples (2 µg) were analyzed by reducing and non-reducing SDS-PAGE using 15 % polyacrylamide gels. The results are shown in figure S2. Under reducing conditions the preparations show a main band of the same size (35 kDa). Under non-reducing conditions a second band with a size of approximately 70 kDa is detected for the variants containing a free cysteine indicating the presence of dimers. Whereas the preparation of CalB-C311A-His contains only very little dimers, the amount of dimers in the CalB-HisGGC preparation is almost 50 %.
Figure S2. Reducing and non-reducing SDS-PAGE of the purified CalB variants. SDS-PAGE under reducing conditions shows one main band of the same size (35 kDa) for all three variants CalB-His (wt), CalB-HisGGC (GGC) and CalB-C311A-His (C311A). The preparation of CalB-C311A-His contains a small amount of other proteins. Whereas the CalB-His preparation again shows only one band under non-reducing conditions, a second band appears for the variants containing the free cysteine (GGC and C311A). The size of the second band is approximately 70 kDa indicating that the preparations contain a certain amount of dimers. Dimerization occurs with a higher probability in the CalB-HisGGC preparation.
Activity measurements in solution To compare the activity of the different CalB variants para-nitrophenol butyrate (p-NPB; Sigma, Taufkirchen, Germany) was used as described previously.[2] Briefly, a p-NPB stock solution was prepared with a concentration of 20 m M in isopropanol. The assay mixture contained 50 m M sodium phosphate pH 7.0, 150 m M NaCl, 0.5 % Triton X-100, 5 % isopropanol. The following substrate concentrations were used: 1000 µM, 200 µM, 80 µM and 50 µM. The reaction was started through adding the respective lipase variant to a final concentration of 100 nM. The lipase variants were preincubated with a 50 fold excess of TCEP in order to ensure that only monomers were present during the measurement. The temperature was kept constant at 25 °C. The formation of the product could be followed through measuring the increase in absorbance every 10 s for a total time of 300 s. All measurements were carried out in triplicate. Assay mixtures containing BSA instead of lipase were used as reference samples. The increase in absorbance of the BSA samples was subtracted from the values obS4
tained from the lipase samples. These corrected curves were used to calculate the reaction velocity v, which describes the concentration of the generated product per minute. The data was fitted with the Michaelis-Menten equation to obtain the respective constants. The results are summarized here in Table S1
CalB-His specific activity [µmol min-1 mg-1] kM [µM] vmax [µM min-1] kcat [min-1] k cat /k M [min-1 µM- 1]
6.96 2807
CalB-HisGGC 5.97 3624
91.6 916
93.9 958
0.326
0.264
CalB-C311A-His 7.32 2331 60.9 858 0.368
Immobilization of the lipase variants Commercially available amino functionalized glass slides (Nexterion Slide A, PEQLAB, Erlangen, Germany) were incubated in 50 m M sodium borate buffer pH 8.5 for 1 h and dried under a stream of N2. This step was necessary to deprotonate the amino groups on the surface, which is essential to achieve coupling to N-hydroxysuccinimide (NHS) activated carboxy groups. The amino surface was converted to a sulfhydryl reactive surface by using NHSPEG-maleimide with a molecular weight of 3400 g mol-1 (Nektar, Huntsville, Alabama). The PEG was dissolved in borate buffer in a concentration of 50 m M. A volume of 200 µL of the PEG solution was incubated on the slide under a 24 mm x 60 mm cover slip in a humid chamber. After 1 h the cover slip was removed and the slide was rinsed with ddH2O and dried with N2. Whereas the slides were incubated with the PEG solution, lipase samples were reduced using TCEP beads (Perbio Science). This step was carried out to obtain free cysteines on the lipases for immobilization to the maleimide-activated surface. The beads were equilibrated with coupling buffer by washing them 3 times with 1 mL buffer. Finally, 10 µL of the beads were mixed with 10 µl of the respective lipase solution (0.6 mL mL-1). After shaking these mixtures for 1 hour at 4 °C the beads were removed by centrifugation. Four different spotting solutions were prepared for each of the lipase variants CalB-His, CalB-HisGGC and CalB-C311A-His: The reduced samples were diluted either with coupling buffer or with coupling buffer containing free cysteine. In addition non-reduced lipases were diluted in both buffers. The final concentration of the lipases in the spotting solution was apS5
proximately 150 µg mL-1 or 4.5 µM. The spotting solutions containing free cysteine exhibit a concentration of cysteine 45 m M, which represents an excess of 10 000-fold. The solutions were spotted onto the maleimide-activated slide using the scheme of Figure 2. Volumes of 2 µL were incubated on the slide for 1 h at 4 °C in a humid chamber. The spots were removed by aspiration and the slide was washed 3x 5 min in TBS (50 m M Tris pH 8.0, 15 m M NaCl, 1 m M CaCl2) + 0.4 % bovine serum albumin (BSA). Detection of immobilized lipases To obtain information about the density of the immobilized lipases on the surface the Nterminal FLAG tag was detected using the M1 antibody (see Western-Blot analysis). The antibody was diluted to a final concentration of 4 µg mL-1 in TBS+BSA. A volume of 5 mL was incubated on the slide for 1 hour at room temperature. After washing the slide 3x 5 min in TBS+BSA, a secondary rabbit anti-mouse antibody carrying an AlexaFluor 647 label (1 µg mL-1 in TBS+BSA) was incubated on the slide for 30 min. The slide was again washed 3x 5 min in TBS+BSA. The slide was scanned for fluorescence on the lipase spots using a LS300 scanner (Tecan). Agar plates containing Tween 80 and CaCl2 were prepared to obtain information about the activity of the immobilized lipases. For the preparation of the agar plates the protocol of Suen et al.[4] has been slightly modified: A mixture of 50 m M Tris pH 7.2, 20 m M CaCl2, 2 % (v/v) Tween 80 and 1.8 % (w/v) agar agar was boiled until the agar agar dissolved completely. The mixture was poured into Petri dishes. After gelation the slide containing the immobilized lipases was put up side down on the surface of the agar plate and incubated over night at room temperature. Images were taken using a digital camera. _____________ [1]
A. Krebber, S. Bornhauser, J. Burmester, A. Honegger, J. Willuda, H. R. Bosshard, A. Plückthun, J. Immunol. Methods 1997, 201, 35-55.
[2]
K. Blank, J. Morfill, H. Gumpp, H. E. Gaub, J. Biotechnol. 2006, in press.
[3]
A. Knappik, A. Plückthun, Biotechniques 1994, 17, 754-761.
[4]
W. C. Suen, N. Y. Zhang, L. Xiao, V. Madison, A. Zaks, Protein Eng. Des. Sel. 2004, 17, 133-140.
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