Supporting Information. Sequence Independent Cloning and Posttranslational. Enzymatic Ligation

Transcription

1 Supporting Information Sequence Independent Cloning and Posttranslational Modification of Repetitive Protein Polymers through Sortase and Sfp-mediated Enzymatic Ligation Wolfgang Ott,,, Thomas Nicolaus,, Hermann E. Gaub,, and Michael A. Nash,,,,* Lehrstuhl für Angewandte Physik, Ludwig-Maximilians-Universität München, Munich, Germany. Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität München, Munich, Germany. Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany. Department of Chemistry, University of Basel, 4056 Basel, Switzerland. Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH-Zürich), 4058 Basel, Switzerland. 1

2 Original synthesized DNA-Sequence (5 to 3 ): DNA Sequence 1: GTACCAGGCGTTGGTGTGCCGGGTGTCGGTGTCCCAGGCGTGGGTGTTCCGGGTGTG GGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGTGTTCCTGGTGCTGGTGTTCCGGGC GGCGGTGTTCCGGTTGGTGGCGTTCCGGGTGGCGGT Translated Protein-Sequence (N-Terminus to C-Terminus): Protein Sequence 1: VPGVGVPGVGVPGVGVPGVGVPGVGVPGAGVPGAGVPGGGVPVGGVPGGG PCR modified DNA-Sequence (5 to 3 ): DNA Sequence 2: GTGCCGGGAGAAGGAGTCCCTGGTGTCGGTGTCCCAGGCGTGGGTGTTCCGGGTGT GGGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGTGTTCCTGGTGCTGGTGTTCCGGG CGGCGGTGTTCCGGGGGGTGGCGTTCCGGGTGAAGGA Translated Protein-Sequence (N-Terminus to C-Terminus): Protein Sequence 2: VPGEGVPGVGVPGVGVPGVGVPGVGVPGAGVPGAGVPGGGVPGGGVPGEG 2

3 Sequence Maps were built with SnapGene (GSL Biotech LLC, Chicago, IL, USA) Sequence of original pet28a-vector with aligned Primers: Forward and reverse primer flanking the multiple cloning site of the pet28a vector, and deleting the restriction sites, HIS-tags as well as the thrombin site and the T7-tag (Fig. S1). Figure S1. MCS of pet28a vector with two primers suitable for linearization and insertion of the ybbr- and sortase c-tag. The linear vector serves as starting template for the ELP insertion. DNA Sequence (5 to 3 ): Colored letters represent the annealing region of the forward (green) and reverse (red) primer. TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAA TTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCAT CACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACA GCAAATGGGTCGCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTC GAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGC TGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAA ACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT 3

4 FW backbone LPETGG (Primer 5, Table 1, Main text) ATATATGGTCTCCTGCCGGAAACCGGCGGCTAACTCGAGTAAGATCCGGCTGC REV pet28a (ybbr_bsai) (Primer 12) - Theoretical primer, not used in this study: ATATATGGTCTCAGCCAGTTTAGAAGCGATGAATTCCAGCATGGTATATCTCCTTC 4

5 Sequence of the modified pet28a-vector already containing a ybbr-tag: Figure S2. MCS of modified pet28a vector used in this study already having a ybbr-tag. The linear vector serves as starting template for the ELP insertion. DNA Sequence (5 to 3 ): Colored letters represent the annealing region of the forward (green) and reverse (red) primers. TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTGTAGAAATA ATTTTGTTTAACTTTAAGAAGGAGATATACATATGGACTCTCTGGAATTCATCGCTTC TAAACTGGCTCTGGAAGTTCTGTTCCAGGGTCCGCTGCAGCACCACCACCACCACCA CCCATGGACTAGTGCTAGCTCTACTAAATTATACGGCGACGTCAATGATGACGGAAA AGTTAACTCAACTGACGCTGTAGCATTGAAGAGATATGTTTTGAGATCAGGTATAAG CATCAACACTGACAATGCCGATTTGAATGAAGACGGCAGAGTTAATTCAACTGACTT AGGAATTTTGAAGAGATATATTCTCAAAGAAATAGATACATTGCCGTACAAGAACT AACTCGAGTAAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCT GCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAG GGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT FW backbone LPETGG (Primer 5, Table 1, Main text) ATATATGGTCTCCTGCCGGAAACCGGCGGCTAACTCGAGTAAGATCCGGCTGC 5

6 REV backbone ybbr (Primer 6, Table 1, Main text) ATATATGGTCTCAGCCAGTTTAGAAGCGATGAATTCCAG Cloning Site of Vector A (Fig. 1): Figure S3. Top: Illustration of the linearized plasmid after PCR amplification, Middle: Zoom in of the BsaI-digested 5 -end, Bottom: Zoom in of the BsaI-digested 3 -end. 6

7 PCR Product ELP I (5 to 3 ): Figure S4. PCR Product ELP I (top) with the two corresponding primers (FW ELP I (Primer 1a, Table 1, Main text) ybbr, REV ELP I (Primer 1b, Table 1, Main text)). The bottom figure shows the BsaI-digested fragment. DNA Sequence of ELP I: DNA Sequence 3: TATATAGGTCTCCTGGCTGTGCCGGGAGAAGGAGTCCCTGGTGTCGGTGTCCCAGGC GTGGGTGTTCCGGGTGTGGGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGTGTTCCT GGTGCTGGTGTTCCGGGCGGCGGTGTTCCGGGGGGTGGCGTTCCGGGTGAAGGAGG AGACC 7

8 PCR Product ELP II (5 to 3 ): Figure S5. PCR Product ELP II (top) with the two corresponding primers (FW ELP II (Primer 2a, Table 1, Main text), REV ELP II (Primer 2b, Table 1, Main text)). FW ELP II ybbr (Primer 8, Table 1, Main text) would be necessary if the ELP II fragment should be ligated first to the ybbr-tag on the linearized backbone instead of the fragment ELP I. The bottom figure shows the BsaI-digested fragment. DNA Sequence of ELP II: DNA Sequence 4: TATATAGGTCTCAAGGAGTACCAGGCGAAGGCGTGCCGGGTGTCGGTGTCCCAGGC GTGGGTGTTCCGGGTGTGGGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGTGTTCCT GGTGCTGGTGTTCCGGGCGGCGGTGTTCCGGGGGGTGGCGTTCCGGGTGAGGGTGA GACCATATAT 8

9 PCR Product ELP III (5 to 3 ): Figure S6. PCR Product ELP III (top) with the two corresponding primers (FW ELP III (Primer 3a, Table 1, Main text), REV ELP LPETGG (Primer 3b, Table 1, Main text)). FW ELP III ybbr (Primer 9, Table 1, Main text) would be necessary if the ELP III fragment is to be ligated first to the ybbr-tag on the linearized backbone instead of the fragment ELP I. REV ELP III (Primer 4, Table 1, Main text) is necessary to create a compatible sticky end for the ELP growing reaction after inserting the first three ELP fragments (I, II, III). The bottom figure shows the BsaIdigested fragment. DNA Sequence of ELP III: DNA Sequence 5: TATATAGGTCTCGAGGGTGTACCAGGCGAAGGGGTGCCGGGTGTCGGTGTCCCAGG CGTGGGTGTTCCGGGTGTGGGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGTGTTCC TGGTGCTGGTGTTCCGGGCGGCGGTGTTCCGGGGGGTGGCGTTCCGGGTGAAGGTCT GCCGGAGACCATATAT 9

10 PCR Product ELP IV (5 to 3 ): Figure S7. PCR Product ELP IV (top) with the two corresponding primers (FW ELP III (Primer 3a, Table 1, Main text), REV ELP III (Primer 4, Table 1, Main text)). FW ELP III ybbr (Primer 9, Table 1, Main text) would be necessary if the ELP III fragment is to be ligated first to the ybbr-tag on the linearized backbone instead of the fragment ELP I. REV ELP III LPETGG (Primer 3b, Table 1, Main text) is necessary to create a compatible sticky end for the initial ELP insertion with fragments ELP I, II and III. ELP IV is necessary for the expansion reaction of the ELP insert (ELP IV replaces ELP III). The bottom figure shows the BsaI-digested fragment. DNA Sequence of ELP IV: DNA Sequence 6: TATATAGGTCTCGAGGGTGTACCAGGCGAAGGGGTGCCGGGTGTCGGTGTCCCAGG CGTGGGTGTTCCGGGTGTGGGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGTGTTCC TGGTGCTGGTGTTCCGGGCGGCGGTGTTCCGGGGGGTGGCGTTCCGGGTGAAGGTG GAGACCATATAT 10

11 Insertion site for ELP plasmid (3x 10 pentapepitdes upwards) Figure S8. Illustrating the insertion site for the plasmid linearization reaction. FW backbone ybbr (Primer 7, Table 1, Main text) and REV backbone ybbr (Primer 6, Table 1, Main text) open the plasmid right after the N-terminal ybbr-tag and the first following ELP fragment I. The three following ELP fragments I, II and IV are ligated in between. Linearized PCR product: The construct now has a ybbr-tag at the 5 - and 3 - end and gets cleaved off after BsaI digestion at the 5 end, leaving an ELP I sticky 5 end for the Golden Gate reaction and a ybbr-sticky end at the 3 end. For the linearization reactions of growing ELP constructs the insertion site always remains the same. This means that the structure of the linear backbone is: - 5 ybbr-[elp I-ELP II-ELP III] n -LPETGG-Backbone-ybbR 3 - BsaI Digestion: 5 -[ELP I-ELP II-ELP III] n -LPETGG-Backbone-ybbR 3 - Inserts are added between the sticky ends of the ybbr-tag and the first ELP I fragment. 11

12 Figure S9. Top: Illustration of the linearized plasmid after PCR amplification with the primers FW backbone ybbr (Primer 7, Table 1, Main text) and REV backbone ybbr (Primer 6, Table 1, Main text) and REV backbone ybbr, Middle: Zoom in of the BsaI-digested 5 -end, Bottom: Zoom in of the BsaI-digested 3 -end. 12

13 Cloning efficiencies: Table S1. Overview of cloning efficiencies. Pentapeptide Repeats Number of colonies analyzed with colony PCR/ restriction digestion (*) Number of clones with correct size in colony PCR/ restriction digestion Number of sequencing reactions performed Correct clones (#) Ratio % % % % % % % % % % 60 with Cysteine % (*) Constructs longer than 60 pentapeptide repeats were analyzed by restriction digestion. (#) Wrong results missed 1x ELP Fragment (10 Pentapeptides) 13

14 Gel pictures of the PCR amplified ELP fragments I-IV and the different linearized plasmids used in this study: Figure S10. Gel pictures showing PCR products of insert (top) and linearized plasmid (bottom). Bands with a red arrow are the corresponding linearized plasmids and were isolated via gel extraction to separate them from the side products. 14

15 Exemplary sequence of ybbr-elp 30 -LPETGG: ybbr-tag ELP 1 ELP 2 ELP 3 Sortase c-tag (LPETGG) DNA Sequence 7 (5 to 3 ): ATGGACTCTCTGGAATTCATCGCTTCTAAACTGGCTGTGCCGGGAGAAGGAGTCCCT GGTGTCGGTGTCCCAGGCGTGGGTGTTCCGGGTGTGGGCGTTCCAGGCGTAGGCGTA CCGGGCGCGGGTGTTCCTGGTGCTGGTGTTCCGGGCGGCGGTGTTCCGGGGGGTGGC GTTCCGGGTGAAGGAGTACCAGGCGAAGGCGTGCCGGGTGTCGGTGTCCCAGGCGT GGGTGTTCCGGGTGTGGGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGTGTTCCTGG TGCTGGTGTTCCGGGCGGCGGTGTTCCGGGGGGTGGCGTTCCGGGTGAGGGTGTACC AGGCGAAGGGGTGCCGGGTGTCGGTGTCCCAGGCGTGGGTGTTCCGGGTGTGGGCG TTCCAGGCGTAGGCGTACCGGGCGCGGGTGTTCCTGGTGCTGGTGTTCCGGGCGGCG GTGTTCCGGGGGGTGGCGTTCCGGGTGAAGGTCTGCCGGAAACCGGCGGCTAA Protein Sequence 3 (N- to C-terminus): MDSLEFIASKLAVPGEGVPGVGVPGVGVPGVGVPGVGVPGAGVPGAGVPGGGVPGGG VPGEGVPGEGVPGVGVPGVGVPGVGVPGVGVPGAGVPGAGVPGGGVPGGGVPGEGVP GEGVPGVGVPGVGVPGVGVPGVGVPGAGVPGAGVPGGGVPGGGVPGEGLPETGG Growing ELP constructs had repeats of the ELP 1, 2 and 3 segments inserted in between the ybbr-tag and the following ELP sequence. 15

16 Protein purification and Sfp-mediated labeling: gels with samples from different steps: Figure S11. Gel of purification steps of the ELP 40 (lane 1-8) via ITC with different illumination methods. Lane 9-14 shows the purified ELP constructs. Sfp appears at about 25 kda throughout all lanes. Subset A shows the fluorescence of the Sfp catalyzed labeling reaction of CoA-647 dye to the ybbr-tag containing ELP constructs. Panel B shows the UV activated tryptophan labeling (ELPs not labeled) via Bio-Rad Stain-Free TM technology. C shows the overlay of A (red) and B (blue). 16

17 Negative control confirming native GG-sfGFP on top of SDS-Gel (Figure 2) Figure S12. Negative control for Figure 2 B-D, showing the electrophoresis running behavior of native GG-sfGFP. Sortase in lane 3, 4, 7, and 8 at ca. 18 kda. Sfp in lane 2, 4, 6 and 8 at ca. 25 kda. GG-sfGFP (native) in lane 1-4 at ca 75 kda and GG-sfGFP (denatured) lane 5-8 at ca. 25 kda. 17

18 Sequence of HIS-TEV-GG-sfGFP: HIS 6 -Tag TEV-Site Glycines sfgfp DNA Sequence 8 (5 to 3 ): ATGCACCACCACCACCACCACGGAGAAAACCTGTACTTCCAGGGAGGGGGCGGTAG CAAAGGTGAAGAACTGTTTACCGGTGTTGTTCCGATTCTGGTTGAACTGGATGGTGA TGTTAATGGCCACAAATTTTCAGTTCGTGGTGAAGGCGAAGGTGATGCAACCATTGG TAAACTGACCCTGAAATTTATCTGTACCACCGGCAAACTGCCGGTTCCGTGGCCGAC CCTGGTTACCACCCTGACCTATGGTGTTCAGTGTTTTAGCCGTTATCCGGATCATATG AAACGCCACGATTTTTTCAAAAGCGCAATGCCGGAAGGTTATGTTCAAGAACGTACC ATCTCCTTTAAAGACGACGGTAAATACAAAACCCGTGCCGTTGTTAAATTTGAAGGT GATACCCTGGTGAATCGCATTGAACTGAAAGGCACCGATTTTAAAGAGGATGGTAA TATCCTGGGCCACAAACTGGAATATAATTTCAATAGCCACAACGTGTATATCACCGC AGACAAACAGAAAAATGGCATCAAAGCCAATTTTACCGTGCGCCATAATGTTGAAG ATGGTAGCGTGCAGCTGGCAGATCATTATCAGCAGAATACCCCGATTGGTGATGGTC CGGTTCTGCTGCCGGATAATCATTATCTGAGCACCCAGACCGTTCTGAGCAAAGATC CGAATGAAAAACGTGATCATATGGTGCTGCATGAGTATGTTAATGCAGCAGGTATTA CCCATGGTATGGATGAGCTGTATAAGTAA Protein Sequence 4 (N- to C-terminus): MHHHHHHGENLYFQGGGGSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGK LTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISF KDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQK NGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKRD HMVLHEYVNAAGITHGMDELYK After TEV digestion: GGGGSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVP WPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKF EGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVED GSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKRDHMVLHEYVNAAGITH GMDELYK 18

19 Gel picture of the PCR amplified, linearized and mutated Cysteine-ELP 60 -LPETGG: Figure S13. Gel picture showing the PCR products of the linearized and mutated Cysteine-ELP 60 -LPETGG bands at 67 and 72 C annealing temperature. The red arrows indicate the desired PCR product. 19

20 PCR product (5 to 3 ): Figure S14. Illustration of PCR product before BsaI restriction. The plasmid was linearized with the primers FW ELP N Cys (Primer 10, Table 1, Main text) and REV backbone (Primer 11, Table 1, Main text). Figure S15. Zoom in 5 end of PCR product before BsaI restriction. 20

21 Figure S16. Zoom in 3 end of PCR product before BsaI restriction. Figure S17. Zoom in 5 end of PCR product after BsaI restriction. 21

22 Figure S18. Zoom in 3 end of PCR product after BsaI restriction. The digested ends were filled to blunt ends with Klenow Fragment and ligated with the T4- Ligase as described in the main text (methods). 22

23 Sequence of final Cysteine-ELP 60 -LPETGG: Cysteine ELP 1 ELP 2 ELP 3 Sortase c-tag (LPETGG) DNA Sequence 9 (5 to 3 ): ATGTGCGTGCCGGGAGAAGGAGTCCCTGGTGTCGGTGTCCCAGGCGTGGGTGTTCCG GGTGTGGGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGTGTTCCTGGTGCTGGTGTT CCGGGCGGCGGTGTTCCGGGGGGTGGCGTTCCGGGTGAAGGAGTACCAGGCGAAGG CGTGCCGGGTGTCGGTGTCCCAGGCGTGGGTGTTCCGGGTGTGGGCGTTCCAGGCGT AGGCGTACCGGGCGCGGGTGTTCCTGGTGCTGGTGTTCCGGGCGGCGGTGTTCCGGG GGGTGGCGTTCCGGGTGAGGGTGTACCAGGCGAAGGGGTGCCGGGTGTCGGTGTCC CAGGCGTGGGTGTTCCGGGTGTGGGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGT GTTCCTGGTGCTGGTGTTCCGGGCGGCGGTGTTCCGGGGGGTGGCGTTCCGGGTGAA GGTGTGCCGGGAGAAGGAGTCCCTGGTGTCGGTGTCCCAGGCGTGGGTGTTCCGGG TGTGGGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGTGTTCCTGGTGCTGGTGTTCC GGGCGGCGGTGTTCCGGGGGGTGGCGTTCCGGGTGAAGGAGTACCAGGCGAAGGCG TGCCGGGTGTCGGTGTCCCAGGCGTGGGTGTTCCGGGTGTGGGCGTTCCAGGCGTAG GCGTACCGGGCGCGGGTGTTCCTGGTGCTGGTGTTCCGGGCGGCGGTGTTCCGGGGG GTGGCGTTCCGGGTGAGGGTGTACCAGGCGAAGGGGTGCCGGGTGTCGGTGTCCCA GGCGTGGGTGTTCCGGGTGTGGGCGTTCCAGGCGTAGGCGTACCGGGCGCGGGTGT TCCTGGTGCTGGTGTTCCGGGCGGCGGTGTTCCGGGGGGTGGCGTTCCGGGTGAAGG TCTGCCGGAAACCGGCGGCTAA Protein Sequence 5 (N- to C-terminus): MCVPGEGVPGVGVPGVGVPGVGVPGVGVPGAGVPGAGVPGGGVPGGGVPGEGVPGE GVPGVGVPGVGVPGVGVPGVGVPGAGVPGAGVPGGGVPGGGVPGEGVPGEGVPGVG VPGVGVPGVGVPGVGVPGAGVPGAGVPGGGVPGGGVPGEGVPGEGVPGVGVPGVGVP GVGVPGVGVPGAGVPGAGVPGGGVPGGGVPGEGVPGEGVPGVGVPGVGVPGVGVPG VGVPGAGVPGAGVPGGGVPGGGVPGEGVPGEGVPGVGVPGVGVPGVGVPGVGVPGA GVPGAGVPGGGVPGGGVPGEGLPETGG 23

24 MALDI-TOF analysis of ELP 30, ELP 40 and ELP 50 For further analysis, three ELP samples were sent to MALDI-TOF analysis to check their grade of purity. Figure S19. MALDI-TOF Analysis of ELP 30 (A, Da), ELP 40 (B, Da) and ELP 50 (C, Da). 24

25 SDS-Gel of Bioconjugation Reactions: Figure S20. An SDS gel picture confirming Sfp-mediated ELP conjugation to a CoA-647 dye (Lanes 1-6; ELP 10 - ELP 60 ). Lanes 7 and 8 of the gel show the result of the Alexa 647 -maleimide-dye bioconjugation to the cysteine-elp 30 (7) and cysteine-elp 60 (8). It was not possible to label the cysteine-elps with the CoA-647 dye, since it did not carry a ybbr-tag anymore. Vice versa, the Alexa 647 -maleimide dye did not label the standard ybbr-containing ELPs, which do not have a cysteine (data not shown). Red is the dye fluorescence (excitation: 530/28 nm and emission: 695/55 nm). The marker lane is shown in blue. 25