A transcription blocker isolated from a designed repeat protein combinatorial library. Shicong Xie 1,$, and Lan Guan 1*

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1 A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen Elena B. Tikhonova, Abdul S Ethayathulla,#, Yue Su,#, Parameswaran Hariharan, Shicong Xie,$, and Lan Guan * Department of Cell Physiology & Molecular Biophysics Center for Membrane Protein Research Texas Tech University Health Sciences Center, Lubbock, Texas * Corresponding Author: Lan Guan, 360 4th Street, 5A63, STOP 655, Lubbock, Texas Telephone: (806) ; Fax: (806) ; and Lan.Guan@ttuhsc.edu # These authors contribute equally. $ Current address: Computational and Systems Biology Graduate Program, MIT, Cambridge, MA Supplementary Information Contents: Supplementary Table S Supplementary Figures S, S2 Supplementary Note

2 Supplementary Table S. Construction efficiency Plasmid Test Sample number N5C N4C N0C Clone with errors in randomized framework position position Restored N5Cm N4Cm N3Cm pinitial Ligation reaction 47 (%) 20 (42.55) 2 (4.26) (2.3) 8 (7.02) 6 (34.04) 2 (44.68) 3 (6.38) pinitial Plasmid library 2 50 (%) 25 (50) 2 (4.00) (22.00) 2 (24.00) 7 (34.00) 4 (8.00) 2.00) pcs9/fx Plasmid library 49 (%) 23 (46.94) 2 (4.08) 8 (6.33) 6 (32.65) 24 (48.98) Total 46 (%) 68 (46.58) 6 (4.) (0.68) 27 (8.49) 44 (30.4) 62 (42.47) 7 (4.79) (0.68), a ligation reaction mixture was used for transformation. 2, a cell-amplified plasmid library was used for transformation. 2

3 Supplementary Fig. S. In vivo functional screen using melibiose fermentation on MacConkey agar plates. a, Examples of primary screen on MacConkey agar plates. E. coli Tuner cells were transformed with a pool of pcs9/n5c plasmids and plated on the lactose-free MacConkey agar plates supplemented with 00 μg/ml ampicillin, 30 mm melibiose, 0. mm IPTG. Most cells form red colonies as Tuner cells without a plasmid (data not shown). Blue arrows indicate the colonies with reduced color; left panel, a halo colony with red color in the center; middle panel, pink colony; right panel, yellow colony. b and c, Patches showing melibiose or glucose fermentation by cells carrying an ANK-N5C clone. Cells with pcs9/et (vector control) or pcs9/n5c-62 (protein control) are also indicated. Clones with reduced melibiose fermentation are labeled in white. 3

4 Supplementary Fig. S2. H-bonding interactions between the position- and -5. The distance (Å) between the main-chain nitrogen at position-5 and the negatively charged carboxyl group of Asp at the conserved position- are indicated. a, ANK-N5C-37. Pro38 in the internal repeat (IR) IV disturbs the H-bonding interactions that are observed in other repeats. b. ANK-N5C- 28. The β-turn of internal repeat V is deformed, and Pro7 is far from the Asp at position-. A new salt-bridge interaction is formed between the randomly selected Glu33 (internal repeat III) and consensus Arg99 (internal repeat V). 4

5 Supplementary Note SI Note Materials. All oligodeoxynucleotides were synthesized by Integrated DNA Technologies. A codon trimer mix solution was custom-prepared by Glen Research. All molecular biology reagents were purchased from New England Biolabs. Vector construction The plasmids used in this study are listed in Table. For the fragment-exchange cloning method (FX) 38, two compatible plasmids pcs9/fx and pacyc/fx were created, all containing a ccdb cassette flanked with two SapI sites. Construction of pcs9/fx. The ANK-N5C#6 construct was cloned by NcoI and HindIII sites into an available plasmid pcs9 derived from an expression vector pqe60 (Qiagen) 52. Two SapI sites were inserted at both ends of the ANK-N5C#6 gene by site-directed mutagenesis. The resulting sequence (5 -aacc ATG GCT CTT CG A GT-3 ) at the 5 end of the gene is compatible with a SapI-processed DNA fragment carrying a 5 overhang of AGT followed by c and an open-reading-frame, yielding a sequence 5 -aacc ATG GCT CTT CGA GTc. Another SapI site was inserted right after the stop codon TAA and blocked the HindIII site. The resulting sequence (5 -TAA gca tgaagagc tgag-3 ) is compatible with an insert fragment carrying a 5 overhang (3 -CGT-5 in the antisense strand) at the 3 end generated by SapI treatment. For a counter-selection, the ANK-N5C#6 gene was replaced with a fragment containing ccdb cassette from the plasmid pinitial 38 ; the construct was maintained in the ccdb survival strain E. coli DB 3. with ampicillin selection. Restriction digestion profile and DNA sequencing analysis confirmed the insertion of the 0.67-kb ccdb cassette. The third SapI site at position 27 of the 5

6 vector was removed by QuikChange site-directed mutagenesis. The resulting cloning/expression vector (5.4 Kb) under IPTG induction was re-named pcs9/fx for construction of the library. Construction of pacyc/fx. Two SapI sites were inserted into both ends of the lacy gene carried by a plasmid pacyc/c6 lacy 54, which is compatible with pcs9-derived constructs for co-expression in E. coli cells. The 0.67-kb ccdb cassette from pcs9/fx was used to replace the lacy gene, yielding pacyc/fx. Construction of plasmid libraries. To construct the pinitial/ank-n5c combinatorial library, the sense primer P-44 (5 -TGGGC GCTCTTC T AGT ATGGATATCG-3 ) and the antisense primer P-45 (5 -ggtggccgctcttcatgctta GTG AT-3 ) were used to assemble/amplify the full-length DNA fragments. To construct the pcs9/ank-n5c combinatorial library, the sense primer P-87 (5 -atatgctcttcg A GTC GAT ATC GGT A-3 ) and the antisense primer P-45 (5 -ggtggccgctcttcatgc TTA GTG AT-3 ) were used, which generate additional stretch of residues (Met-Ala-Leu-Arg-Val) prior to the Asp-Ile-Gly of ANK-N5C proteins. After FX-cloning reactions 38, a portion of a transformation reaction was supplemented with 0- ml fresh Luria-Bertani (LB) broth containing a selection antibiotic and shaken at 37 C overnight for plasmid isolation. To separate the library clones, the isolated plasmid pool was transformed into E. coli XL-Blue competent cells or E. coli Stellar TM chemically competent cells. The remaining portion of the transformation reaction was plated onto antibiotic-selection plates for counting ligation/transformation efficiency. About 50 plasmid clones from each condition were randomly selected for DNA sequencing analysis (Supplemental Information Table S). 6

7 Construction of pacyc vectors encoding MelB, MelAB, MelR, or LacY. The genes melab and melr from the chromosomes isolated from the Tuner strain and the genes melb and lacy from plasmids pt40/melb Ec -6His 5 and pt75/lacy-0his 53 were PCR-amplified and cloned into the pacyc/fx vector, respectively, yielding non-his-tagged plasmids pacyc/melb, pacyc/melab, pacyc/melr, and pacyc/lacy for functional studies. To prepare the plasmid pcs9/et without ccdb gene for control, a plasmid pcs9/ank-n5c-62 was digested with SapI, blunted with T4 DNA polymerase, and re-ligated by T4 DNA ligase. Another control plasmid pacyc/et without ccdb gene was obtained unexpected. Protein overexpression To facilitate protein purification, ANK-N5C clones were cloned into the overexpression vector p7xc3h 38 with kanamycin resistance by the FX cloning method, which should generate Met- Ser-Met at their N-terminus. MelA was cloned into the overexpression vector p7xnh3 38 with kanamycin resistance by the FX cloning method. E. coli T7 Express cells containing a given plasmid were grown in LB broth with 40 mg/l kanamycin in a 37 C shaker. The overnight cultures were diluted to 5% with fresh LB broth with 40 mg/l of kanamycin, incubated at 37 C until absorption at 600 nm reached about 0.6. Protein expression was induced by 0.5 mm IPTG at 30 C for 4-5 h. Purification of MelA. Cells were harvested, suspended in a buffer containing 50 mm NaP i (ph 7.5), 200 mm NaCl, 5% glycerol, and mm PMSF, and broken by passage through an Emulsiflex at 0000 psi, followed by high-speed centrifugation using a Beckman JA8 rotor at 37745g for 30 min. The His-tagged MelA protein was purified by cobalt-affinity chromatography using Talon resin (Clontech). Washed with 30 mm imidazole, MelA proteins were eluted using 200 mm imidazole-containing buffer. The elution fractions were concentrated 7

8 using VIVASPIN 20 (30K MWCO, Millipore) and dialyzed against 2 x -L dialysis buffer consisting of 20 mm Tris-HCl (ph7.5), 00 mm NaCl, and 0 % glycerol. Purification of ANK-N5C-37 and ANK-N5C-28. The purification was carried out following protocols described in MelA purification except for using 5 mm imidazole for washing. A VIVASPIN concentrator of 5K MWCO was used for concentration. Overexpression and purification of MelB. The plasmid pk95 ΔAHB carries a full-length MelB of E. coli (MelB Ec ) with a 6His tag at the C-terminus was used for overexpression in E. coli DW2 strain 5. Cell growth, membrane preparations, and protein purification were carried out as described 42. Preparation of membrane fractions for Western blot analysis Membrane proteins were fractionated by ultracentrifugation as described 39. Protein concentration assay Protein concentration of cell lysates or membrane fractions was estimated by Bio-Rad Protein assay (Bio-Rad) for Western blot analysis. Concentrations of ANK-N5C, MelA, and MelB proteins were measured with Micro BCA TM Protein Assay Kit (Thermo Scientific). BSA was used as standard for both methods. 8