ononononociono □onononononon ODODODOnODODO nonononononon o □ o □ o " ~ " o □ o □ o JJIj Kód předmětu: C8980 I UfU ^ MASARYKOVA UNIVERZITA OnOnOu^uOnOnO □onononononon ODODODOnODODO Protein expression and purification • IV. DNA cloning Lubomír Janda, Jozef Hritz, Blanka Pekarové, Radka Dopitová and Adam Norek Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. HHf £5 Cti ^S^^fc I MINISTERSTVO ŠKOLSTVÍ, OP Vzdělávání EVROPSKÁ UNIE ^0 ■ pro konkurenceschopnost -M jya ^ INVESTICE DO ROZVOJE VZDĚLÁVÁNÍ DNA cloning 4.1. Introduction: correctness of your construct - cloning strategy ATGGGCGGCATccACAGGGTGAACAGATGTACCGGAGGGTGTATCGTCTGCATGAGCGCCTGGTAGCCATCCGCACTGAGTACAACCTCC GGCTGAAGGCAGGAGTGGGTGCCCCTGTGACCCAGGTGACCCTGCAGAGTACACAGAGGCGCCCAGAGCTAGAGGACTCCACACTGCGCT ACCTGCAAGACCTGCTGGCCTGGGTAGAGGAGAACCAGCGTCGAATAGACAGTGCTGAGTGGGGCGTGGACTTGCCCAGTGTGGAGGCCC AGCTGGGCAGCCACCGAGGCATGCATCAGTCTATAGAGGAATTTCGGGCCAAGATCGAGCGGGCTCGGAATGATGAGAGCCAGCTCTCCC CTGCCACCCGGGGTGCCTACCGGGACTGCCTAGGTCGCCTAGACCTGCAGTATGCAAAGCTGCTGAACTCCTCCAAGGCCCGCCTCCGGT CCCTGGAG^ nn GGAGTGACC t AGATCCAGi5 GGAGCTGGi5 CGGAGGAAC TGCAGGATG TGAAGCCAC AGGGTGACC TGTGCTTTC ACCAGCTTC TCCGCACAC GTGGCTTTG AGGGTGAGC -,r,rrr,r,r,r,r,r,7xr,r,r EJ H < 0 b 1 u u b 0 i ■ i—i 1 o O O u u "~> >r. OS o-\ I I M I_, 41 'i\r,r,i\i\r,r,i\rT,Tr,i\' U ,r,rrr,r,r,rrr,7N7Nrrr,7Nr,7N7N7Nr,7N7Nr,7Nr,r,7Nr,r,7N7Nr,r u b O °i S-i ■< u se r.1 i-H II 2 S - « ^ □ 6 q «3 Cq w o 4 S ^ 2S N U ■ " rv« i—i -i 1 ■ SB > ><; &q cq t>5 '^ggctttgatt \aaatcaagg :agacacagt sttcgggagg SAGgATCTGC \TTGTGCAGT \cTGTGCACA stgccttctg \cactgtggc :tagtcacgt _ :aggatgccg 3 \gcctggagc DGVRANELQLRWQEYRELVLLLLQWIRHHTAAFEERKFPS SFEEIEILWCQFLKFKETELPAKEADKNRSKVIYQSLEGAVQAGQLK IPPGYHPLDVEKEWGKLHVAILEREKQLRSEFERLECLQRIVSKLQMEAGLCEEQLNQADALLQSDIRLLASGKVAQRAGEVERDLD KADGMIRLLFNDVQTLKDGRHPQGEQMYRRVYRLHERLVAIRTEYNLRLKAGVGAPVTQVTLQSTQRRPELEDSTLRYLQDLLAWVE ENQRRIDSAEWGVDLP SVEAQLGSHRGMHQSIEEFRAKIERARNDESQLSPATRGAYRDCLGRLDLQYAKLLNSSKARLRSLESLHG LQLCCCIEAHLKENTAYFQFFSDVREAEEQLQKLQETLRRKYSCDRTITVTRLEDLLQDAQDEKEQLNEYKGHLSGLAKRAKAIVQL VEECQKFAKQYINAIKDYELQLITYKAQLEPVASPAKKPKVQSGSESVIQEYVDLRTRYSELTTLTSQYIKFISETLRRMEEEE Key concepts: • Knowing the objectives before DNA cloning • Appreciating the complexity of plasmid systems in terms of the numbers of distinct plasmid options 4.2. The key questions before DNA cloning 4.2.1. DNA-protein analysis 4.2.1.1. Plasmid map DNA sequence ATGGCTAGCACAGATTCAGAGAGTGAGACTAGGGTCAAGTCAGTGCGTACCGGTCGAAAG CCTATTGGGAACCCAGAGGACGAGCAAGAGACTTCCAAGCCGAGTGACGATGAATTCTTA AGAGGAAAGAGAGTTCTTGTGGTCGATGATAACTTTATATCACGTAAAGTTGCAACAGGA AAGCTGAAGAAGATGGGAGTCTCAGAGGTCGAACAATGCGACAGTGGGAAAGAAGCTTTG AGATTAGTCACTGAAGGGCTTACACAAAGAGAAGAACAAGGTTCAGTAGATAAACTTCCG TTTGACTACATATTCATGGACTGCCAAATGCCAGAAATGGATGGCTATGAAGCAACTAGA GAGATTAGGAAAGTGGAGAAAAGTTATGGGGTGCGTACACCAATTATAGCTGTATCTGGT CATGATCCTGGTTCAGAGGAAGCAAGAGAAACCATTCAAGCTGGAATGGACGCCTTCTTA GATAAAAGC T T GAAT CAAC T T GCAAAC GT CAT TAGAGAAAT C GAAAGCAAAC GT CAC www.expasy.ch translate MASTDSESETRVKSVRTGRKPIGNPEDEQETSKPSDDEFLRGKRVLVVDDNFISRKVATG KLKKMGVSEVEQCDSGKEALRLVTEGLTQREEQGSVDKLPFDYIFMDCQMPEMDGYEATR EIRKVEKSYGVRTPIIAVSGHDPGSEEARETIQAGMDAFLDKSLNQLANVIREIESKRH3 4.2. The key questions before DNA cloning 4.2.1. DNA-protein analysis 4.2.1.2. Secondary structure prediction www.expasy.ch jpred3 mastdsesetrvksvrtgrkpignpedeqetskpsddeflrgkrvlvvddnfisrkvatg —eeeeee----eeeeeeeeee-----------------.-^-'--eeeeee—hhhhhhhhh KLKKMGVSEVEQCDSGKEALRLVTEGLTQREEpG^vdklpfdyifmdcqmpemdgyeatr hhhh----eeeee—hhhhhhhhhh-------------eeeeee------hhhhhh eirkveksygvrtpiiavsghd^@seearetiqagmdafldkslnqlanv.jreieskrh hhhh---------eeeee^--—hhhhhhhhhh-----e---HHtJHtfTHHHHHHHH--- .................. 4.2.1.3...EK5mains detected by SMART _________ .............. www^pasy.ch SMART ......... ...«*-—— ........ ^vlvvddnfisrkvatgk^kkMgvseveqcdsgkealrlvtegltqreeqgsvdklp fdyifmdcqmpemdg.¥-e^treirkveksygvrtpiiavsghdpgseearetiqagmda FLDKSLNQLANVl"* Confidently predicted domains, repeats, motifs and features: Name Begin End E-value REC 43 171 1.19e26 4.2.1.3. Domains detected by SMART Histidine kinase from doména Three constructs of receiver domain. pET 28 2 3; 4 5 6i 7 S m 0123 11111 4' 5 6 7 8 212 2 : 0 1 2 3 4 5 6 7! 8 9 0 l! DNA cloning Key concepts: • Knowing the objectives before DNA cloning • Appreciating the complexity of plasmid systems in terms of the numbers of distinct plasmid options 4.2. The key questions before DNA cloning 4.2.2. Plasmid option 4.2.2.1. Plasmid map ap polylmker rmB terminator DNA cloning 4.2. The key questions before DNA cloning 4.2.2. Plasmid option 4.2.2.1. Plasmid map Strong promoter Promoter regulation Transcription terminator Ribosome binding site SD-AUG spacing and base composition A. Expression clone structure: ptac, ptrp, Xpi, pT7 ptrp-tryptophan/IAA ptac-IPTG Xpl- temperature pT7-IPTG T7 term, rrnTl,T2 AAGG (upstream of the AUG initiation) Spacing is crucial to high level expression. (optimal distance 6-10 bp, AT rich base composition) —► attB\ ^——^ atmi (promoter) m rbs_aJP Open Reading Frame Stop B. Expression clone sequence: Shine-Dalsamo Kozak Open reading frame (amino end) 5' - ACA AGT TTG TAC AAA AAA GCA GGC TTC'GAA GGA GAT AGA ÄCC? ATG NNN NNN NNN — 3' - [TGT TCA AAC ATG TTT TTT CGT CCG AJAG GTT CCT CTA TCT TGG TAC NNN NNN NNN — arrBl Translation start* Open reading frame (carboxy end) — NNN NNN NNN JAG GAC CCA GCT TTC TTG TAC AAA GTG GT - 3' — NNN NNN NNN ATC CTG GGT CGA AAG AAC ATG TTT CAC CAI - 5' Translation stop a??B2 4.2. The key questions before DNA cloning 4.2.2. Plasmid option 4.2.2.2. Promoters locilV5, toe and trc promoters are repressed by the lac repressor (loci or loclq) and induced with IPTG. Trp promoter is repressed by the trp repressor and induced with tryptophan (or indole-3-acetic acrylic acid). 77 promoter requires expression of phage RNA polymerase (host strain usually contains this polymerase expressed from lac UV5 promoter induced by addition of IPTG). Pl lambda phage promoter exhibits maximum expression when induced and has low basal expression when the cl repressor is present. 8 4.2. The key questions before DNA cloning 4.2.2. Plasmid option 4.2.2.2. Promoters 4.2.2.2.1. T7/ac promoter Relative basal uninduced expression levels of cloned P-galactosidase with various vector/host combinations • Promoter T7 T7 T7 11 lac 11 lac lilac • Host (DE3) (DE3) (DE3) (DE3) (DE3) (DE3) Activity 100% pLysS 30% pLysE 10% 10% pLysS 3% pLysE 1% 10 4.2. The key questions before DNA cloning 4.2.2. Plasmid option 4.2.2.3. Examples of E. coli expression systems Vector Promoter/ system_induction method Special host protein tag strains required: Source Web site Pinpoint toc/IPTG or 77 I PTG Yes pET 77 I PTG Yes pGEX toc/IPTG No pBAD oro BAD Yes pLEX Pi/trp Yes pPROTet Paet/anhydrotetracyclin No pTYB 77 I PTG Yes pMAL toc/IPTG Yes pQE 75/1PTG Yes/TOPP pCAL 77/1 PTG Yes pLAG toc/IPTG Yes Biotin binding domain www.promeRa.com His6, T7 gene 10 www.novaRen.com GST www.amershambiosciences.com His6, GFP His, www.invitroRen.com www.clontech.com Chitin binding domain www.neb.com Maltose binding domain His, www.qiaRen.com Calmodulin binding www.stratagene.com peptide www.sigmaaldrich.com pUbEx15 tac/IPTG Yes ubiquitin janda@vri.cz DNA cloning 4.2. The key questions before DNA cloning 4.2.3. N-terminal amino acids N-terminal amino acids that reduce stability of proteins. N-degrons • Phe, Leu, Trp, Tyr and Arg, Lys, Tobias et al, 1991, Science; Humbard et al., 2013, JBC Amino acids stabilized in penultimate position N-terminal methionin. His, Gln,Glu, Phe, Met, Lys, Tyr, Trp, Arq Hirel et. al., 1989, PNAS; Lathrop et al. 1992; Liao et.al., 2004, Protein Science Methionione aminopeptidase remove the initiator Met in proteins when the second residue is Glv, Ala, Sen Cvs, Thr, Pro or Val Bonissone et al., 2013, Molecular and Cellular Proteomics 12 DNA cloning 4.2. The key questions before DNA cloning 4.2.4. Protease recognition sites Check the sequence of the fusion partner for the presence of additional protease recognition sites. Pro-Arg/Gly Pro-Lys/Leu Ala-Arg/Gly Gly-Lys/Ala lle-Arg/Ser Leu-Arg/Ala lle-Arg/lle Leu-Glu-Val-Leu-Phe-Gln/Gly-Pro Thrombin pH 8.0 PreScission pH 8.9 Factor Xa pH 6.5-7.5 Enterokinase pH 7.0-8.0 TEV protease pH 5.5-8.5 lle-Glu-Gly-Arg/X Asp-Asp-Asp-Asp-Lys/X Glu-Asn-Leu-Tyr-Phe-Gln/Ser 14 4.2. The key questions before DNA cloning 4.2.5. Antibiotic selection bla gene ampicillin resistance Ampicillin x Carbenicilin kan gene kanamycin resistance 4.2. The key questions before DNA cloning 4.2.6. Codons with translation problems Arginine agg BL21-Codon plus-RI Isoleucine Leucine Glycine Proline aga cga cgg aua cua gga 4.2. The key questions before DNA cloning 4.2.6. Codons with translation problems Leu-CUA lle-AUA Pro-CCC Gly-GGA http ://www. kaz u sa. o r. i p/cod o n/ Escherichia coli K12 Arabidopsis thaliana uuu 19 7 UCU 5 7 UAU 16 8 UGU 5 9 UUU 21 8 UCU 25 2 UAU 14 6 UGU 10 .5 uuc 15 0 UCC 5 5 UAC 14 6 UGC 8 o uuc 20 7 UCC 11 2 UAC 13 7 UGC 7.2 UUA 15 2 UCA 7 8 UAA stop UGA stop 1 UUA 12 7 UCA 18 3 UAA stop UGA stop UUG 11 9 UCG 8 0 UAG stop UGG 10 UUG 20 9 UCG 9 3 UAG stop UGG 12 . 5 CUU 11 9 ecu 8 4 CAU 15 8 CGU 21 1 CUU 24 1 ecu 18 7 CAU 13 8 CGU 9.0 cue 10 5 6 CAC 13 1 CGC 26 o cue 16 1 CCC 5 3 CAC 8 7 CGC 3.8 CUA 5 3 CCA 6 6 CAA 12 1 CGA 4 3 CUA 9 9 CCA 16 1 CAA 19 4 CGA 6.3 CUG 46 9 CCG 26 7 CAG 27 7 CGG 4 1 CUG 9 8 CCG 8 6 CAG 15 2 CGG 4 . 9 AUU 30 5 ACU 8 0 AAU 21 9 AGU 7 2 AUU 21 5 ACU 17 5 AAU 22 3 AGU 14 . 0 AUC 18 2 ACC 22 8 AAC 24 4 AGC 16 6 AUC 18 5 ACC 10 3 AAC 20 9 AGC 11 . 3 AUA 3 7 ACA 6 4 AAA 33 2 AGA 1 4 AUA 12 6 ACA 15 7 AAA 30 8 AGA 19.0 AUG 24 8 ACG 11 5 AAG 12 1 AGG 1 6 AUG 24 5 ACG 7 7 AAG 32 7 AGG 11 . 0 GUU 16 8 GCU 10 7 GAU 37 9 GGU 21 3 GUU 27 2 GCU 28 3 GAU 36 6 GGU 22 .2 GUC 11 7 GCC 31 6 GAC 20 5 GGC 33 4 GUC 12 8 GCC 10 3 GAC 17 2 GGC 9.2 GUA GUG 11 26 5 4 GCA GCG 21 38 1 5 GAA GAG 43 18 7 GGA 9 2 GUA GUG 9 17 9 4 GCA GCG 17 9 5 0 GAA GAG 34 32 3 GGA 24 .2 4 GGG 8 6 J 2 GGG 10.2 5.3/7.2/9.9 3.7/7.5/12.6 9.8/5.3 9.2VI6&/24.2 Arg-CGA Arg-CGG Arg-AGA Arg-AGG 4.3/6.2/6.3 4.1/11.4/4.9 ■> 1.4/12.2/19.0 ■> 1.6/12.0/11.0 17 Key concepts: Being aware of solubility as a function of protein structure 4.3. Protein solubility http://www.biotech.ou.edu/ • Low solubility in aqueous solvents is often regarded as an indication that a protein is "hydrophobic". • As native, properly folded structures aggregate less than unfolded, denatured ones, there is a close relationship between solubility and stability. • The free energy of protein stabilization in an aqueous solution is very low (12 kcal/mol at 30°C). • Free energy of unfolding is observed to be only 5-20 kcal/mol. • Consequently, proteins are on the verge of denaturation. DNA cloning SolubllUy of p - laetuglobulin at various [NaCI] 1 2.0 E 3 1.0 .001 M 5.0 5.2 5.4 5.6 pH Most precipitation ppj" > sol- > cocr > cr NHt > K+ > Na+ Least precipitation Isoelectric focusing gives the pi, the pH at which the protein shows no net charge in isoionic conditions. Generally, charged proteins can be "salted in" by counterions. 19 DNA cloning 4.3. Protein solubility 4.3.2. Determining hydrophobicity http://www.roselab.jhu.edu/~raj/MISC/hphobh.html 20 DNA cloning 4.3. Protein solubility 4.3.3. Solubility model http://www.biotech.ou.edu/ The revised Wilkinson-Harrison solubility model CV =A,1hT) + X21 C™-'- 0.03) n number of amino acids in the protein N, G, P, S number of Asn, Gly, Pro, or Ser residues R, K, D, E number of Arg, Lys, Asp, or Glu residues ^, X2 coefficients (15.43 and -29.56) The probability of the protein being soluble is based on the parameter CV - CV, where CV is the discriminant, equal to 1.71. If CV - CV is positive, the protein is predicted to be insoluble, while if CV - CV is negative, the protein is predicted to be soluble. The probability of solubility or insolubility can be predicted from the following equation: Probability of solubility or insolubility = 0.4934 + 0.276 |(CV-CV')| - 0.0392 (CV-CV')221 DNA cloning 4.3. Protein solubility 4.3.4. Protein engineering to increase solubility 4.3.4.1. Amino acid solubility and water affinity • Hydrophobic amino acids cluster to avoid water. • Most positively charged and amide side chain residues (His, Lys, Arg, Gin, Asn) were on the surfaces of the proteins studied. • The interiors were primarily composed of aliphatics (Gly, Ala, lie, Leu, Val, Phe). • But only 23% of Trp residues and 13% of the Tyr in the structures were not accessible to the solvent, similar to that of the negative polar residues Glu (20%) and Asp (14.5%). Amino acid Transfer free energy kJ/mol % buried Phe F 15.5 48% | Met M 14.2 lie I 13 65% | Leu L 11.7 41% | Val V 10.9 56% Cys C 8.4 47% Trp W 7.9 23% [aft A 6.7 38%] Thr T 5 25% Gly G 4.2 37% | Ser S 2.5 24% Pro P -0.8 24% Tyr Y -2.9 13% His H -12.5 19% Gin Q -17.1 6% Asn N -20.1 10% Glu E -34.3 20% [Lys K -36.8 Asp D -38.5 15% |Arg R -51.4 0%] 22 4.3. Protein solubility 4.3.4. Protein engineering to increase solubility 4.3.4.2. Peptide solubility • For peptides of more than 8 amino acids, sequences favouring oc-helix or random coil structures are more soluble in polar solvents than those forming p-sheet structures. • For other peptides, insertion of arg-N02 residues, or replacement of hydrophobic residues, improved solubility and lowered aggregation tendencies. Amino acic 1 Transfer free energy Chou-Fasman kJ/mol % buried coil index Phe F 15.5 48% 0.71 Met M 14.2 ■ 0.58 lie 1 13 0.66 Leu L 11.7 41% 0.68 Val V 10.9 56% 0.62 Cys C 8.4 47% 1.18 Trp W 7.9 23% ■■ Ala A 6.7 38% Thr T 5 25% 1.07 Gly G 4.2 37% 1.5 Ser S 2.5 24% 1.82 Pro P -0.8 24% 1.59 Tyr Y -2.9 13% 1.06 His H -12.5 19% 1.06 Gin Q -17.1 6% 0.86 Asn N -20.1 10% 1.35 Glu E -34.3 20% 1.2 Lys K -36.8 4% 0.98 Asp D -38.5 15% 1.2 Arg R -51.4 0% DNA cloning 4.3. Protein solubility 4.3.4. Protein engineering to increase solubility 4.3.4.3. Primary structure alterations • Replacement of the hydrophobic EGN@GKIIDYIKLMFHHWFG C-terminal amino acids of penicillin-binding protein 5 with a shorter hydrophilic sequence - IRRPAAKLE -made the protein soluble and allowed crystallization. •A 13 residue deletion E VLN E N LLR<0>VA in a-casein makes the molecule more soluble. •Phenylalanine residues are likely to self-interact and are frequently found at subunit interfaces. Amino acid Transfer free energy % buried Chou-Fasman coil index DNA cloning 4.3. protein solubility 4.3.4. Protein engineering to increase solubility 4.3.4.3. Primary structure alterations • A series of point mutations altered the stability and solubility of insulin. Asn21 is deamidated in an acid solution, resulting in a dimer formation with Gly, Ser, Thr, Asp, His, and Arg. • Specific sequence changes in proteins from a thermophilic organism show a tendency to replace lysine and glutamic acid with arginine and aspartic acid and a preference for the hydrophobic amino acids Phe, Val and Me over Leu, Ala and Met. • Most of these changes occur in cc-helical regions and increase the net hydrophobicity of the residue. Amino acic Transfer free energy Chou-Fasman kJ/mol % buried coil index Phe F 15.5 48% 0.71 1 Met M 14.2 0.58 lie I 13 65% 1 0.66 1 Leu L 11.7 41% 0.68 Val V 10.9 56% 1 0.62 ) Cys C 8.4 47% 1.18 Trp W 7.9 23% 1 0.75 ■ Ala A 6.7 38% 1 0.7 ■ Thr T 5 25% 1.07 Gly G 4.2 37% 1.5 Ser S 2.5 24% 1.82 Pro P -0.8 24% 1.59 Tyr Y -2.9 13% 1.06 His H -12.5 19% 1.06 Gin Q -17.1 6%| 0.86 ■ Asn N -20.1 10% 1.35 Glu -34.3 20% 1.2 Lys K -36.8 4% 0.98 Asp D -38.5 15% 1.2 Arg -51.4 0% DNA cloning 4.3. Protein solubility 4.3.4. Protein engineering to increase solubility 4.3.4.4. Post-isolation alterations • One can alter the solubility of isolated proteins in vitro by coupling to polyethylene glycol (Knauf et al., 1988). 4.3.4.5. Designer proteins A site directed mutagenesis might simply replace a surface hydrophobic amino acid with acidic residues when aggregation problems arise. Obviously, the problem of designing soluble proteins is greatly dependent on the ability to predict protein structure. www.expasy.ch Amino acid Transfer free energy Chou-Fasman kJ/mol % Buried coil index Phe F 15,5 48% 0.71 Met M 14,2 50% 0.58 lie I 13 65% 0.66 Leu L 11,7 41% 0.68 Val V 10,9 56% 0.62 Cys C 8,4 47% 1.18 Trp W 7,9 23% 0.75 Ala A 6,7 38% Thr T 5 25% 1.07 Gly G 4,2 37% 1.5 Ser S 2,5 24% 1.82 Pro P -0,8 24% 1.59 Tyr Y -2,9 13% 1.06 His H -12,5 19% 1.06 Gin Q -17,1 6% 0.86 Asn N -20,1 10% 1.35 Glu -34,3 20% 1.2 Lys K -36,8 4% 0.98 Asp D -38,5 15% 1.2 Arg -51,4 0% 1.0Ä 4.4. Gene cloning 4.4.1. Gateway cloning for protein expression The protein encoding by ccdB gene interferes with the activity of DNA gyrase and acts to inhibit partitioning of the chromosomal DNA. Donor Vector (pDonr-223) attPI attP2 attBI PCR Product attB2 GOl f sele attLl attL2 BPCIonase select for Spc resistance attRl attR2 Entry Clone f LR < ^ sel€ Destination Vector Clonase select for Amp resistance attBI attB2 Expression Clone GOi = gene of interest SpcR = spectinomycin resistance gene AmpR = ampicillin resistance gene prom = transcriptional promoter ccdB = toxic negative selection marker CAT = chloramphenicol resistance gene 4.4. Gene cloning 4.4.1. Gateway cloning for protein expression • PCR reaction of the gene containing the terminal att sites • BP reaction of the 1st cloning • Entry clone - entry vector • LR reaction of the 2nd cloning • Destination vector - terminal vector T xi_ NNN //— NNN ACA AGT TTE TAC AAA AAA GCA GGC TNN TGT TCA AAC ATG TTTI TTT CGT CCG ANN From aflFM From Destination — Vector _ iGENE^ From affl.1 From Entry_ Clone h- ^GENE: P A f. 1 Y K V V NNN NIAC CCA GET TT|C TTG TAC AAA GTG GT NNN NTG GGT CGA AAG AAC ATG From affl.2 From Entry Clone N NNN __// T CAC CAIN NNN ~7/ From a«R2 From Destination Vector 28 4.4. Gene cloning 4.4.1. Gateway cloning for protein expression GOI-stop GOI-nonstop Aminoterminal fusions Aminoterminal and/or carboxyterminal fusions Kozak-GOI-stop Aminoterminal fusions or native eukaryotic expression TEV-GOI-stop Cleavable aminoterminal fusions TEV-GOI-TagCleavable aminoterminal fusions with carboxyterminal epitope/purification tag SD-GOI-stop Native expression in E. coli Tag-GOI-stopAminoterminal tag inside the entry clone DNA cloning 4.4. Gene cloning 4.4.2. Flexi vector cloning Ligation-dependent cloning method facilitated by selection for the replacement of a toxic gene insert in an acceptor vector. http://plasmid.hms.harvard.edu Cloning efficiency: Human 98.9% Mouse 98.9% Rat 98.8% C. elegans 98.5% Zebra fish 97.8% Arabidopsis 97.6% Yeast 97% 4.4.3. The polymerase primer extension (PIPE) BAD/T7 SpeedET MGSDKIHHHHHHENLYFQG Primer 1 ccdB STOP-cgcgac-Pac I Kan r 30 DNA cloning 4.4. Gene cloning 4.4.4. In-fusion PCR cloning http://bioinTo.clontech.com/inTusion/ The system is based on an enzyme with proof-reading exonuclease activity that catalyses the joining of DNA duplexes via exposure of complementary single-stranded sequences. 4.4.5. LIC vectors . His tag lac operator T7 promoter \ RBS TEV si ± Insert e LIC site Ssp Leader Sequence Nde I (ATG) Bglll Kpnl Hindlll BamlU T7 terminator 31 IV. DNA cloning -CTGTACTTCCAATCCAAT -GACATGAAGGTTAGGTTA T4 polymerase - -CTG --GACATGAAGGTTAGGTTA ATTGGAAGTGGATAACGG-TAACCTTCACCTATTGCC dGTP ATTGGAAGTGGATAACGG • GCC TACTTCCAATCCAATGCX----TAACATTGGAAGTGGATAA ATGAAGGTTAGGTTACGY----ATTGTAACCTTCACCTATT T4 polymerase dCTP TACTTCCAATCCAATGCX----TAAC CGY----ATTGTAACCTTCACCTATT Annealed (N-terminal side) LYFQSNA------ -- -CTGTACTTCCAATCCAATGCX.....------------ ---GACATGAAGGTTAGGTTAC GY.....------------ 32 DNA cloning 4.4. Gene cloning 4.4.6. High-throughput cloning and protein expression analysis Process Workflow Stage 1: Vector annealing and cell transformation (Prepared with Robots) Stage 2; Plating for individual clone selection (Prepared Manually) Stage 3: Overnight growth @37°C Stage 4: Transfer select colonies into Bacterial growth cultures Stage 5: Remove aliquot as a temporary freezer stock 1. 3. 96 we// plate of Transformed celts 48 well agar Clone selection plates T«0 1 © i 48 Deepwell plates of Bacterial growth cultures / \ / \ 48f>wpwe»l 11 48D*#pvw(l c 4* 0#vpwN «3 48 0**pvwHI «4 \ / \ / 96 well plates of temporary freezer stocks 33 DNA cloning 4.4. Gene cloning 4.4.6. High-throughput cloning and protein expression analysis Stage 6: IPTG addition to growth cultures for induction of protein expression Stage 7: Aliquot removal for protein expression screening Stage 8: Centrifugation of protein expression samples and 48 Deepwell plates of Bacterial growth culture Stage 9; Process all plates for expression and solubility screening Top Tep #1 48 Deepwell #2 4SDeepw0|l M \ / \ 7 7. 96 well plates of protein expression sampfes 8. i i i © i 1 i j Process ail four 48 Deep we// plates of Bacterial gro wth cultures for solubility screening and process all two 96 well plates for protein expression screening OGenScript The Biology CRO innovíDDn Parm*r n Drag DrscovwyJ Cjotal an řř: Gere name: CKIlrd CsLlnizeLl iar e^piesírsr n f- cail rjnů _ wrawsBflíffBprftJ OptimumGene™ Codon &=re eictn 43S i^j r— ^ Op Ara yi s- coidLcLetl cy: J aso n Zho u, Ph. C ArdyEfc crested: DBf24f2011 D1:14:Q6 OptimumGene™ Codon Optimization Analysis James let V732-B85-9ia6 -aracertemlalAye., Fan; 1-732-21WE62 Optlmlzallon Paranie:er-5 opt TiLTiGeie^-aigoriirírn DpiimzEB a ^artety of parameters-irai are critical ta tn& efficiency oralis expnesslar. inclLdhg tut not imibed ;s: Codon us-*ge tli: GCcanlenl Cp5 clnuc ecUd?: co-rivrt mRHA-iccordar^ sItjcIj-c Ci>pll: 'd crfl xltsx PiemB:u-í PofrAxHss Interrd i±l itm and rDowral brelng Utex Neaair.c CpG xlarcx RNA ri:s3ir^ mclH lARE: Rcdcb: s*íL«rít: ■.drutrřJM.: ~evít:-í tcjm: i"id Dj-aa rsg«ti R-5-:1:Uan tbes^ar: l-riř-Tíre-wtfi cfcrlng 35 DNA cloning Results E. coli 1. Codon usage bias adjustment Frequency of Optimal Codons (FOP) 2. GC Content Adjustment i IOC' eo ■ 70 ■ 60 ■ c 50 ' V 4-1 40 c o u 30 ■ u in 10 ■ 0 GC Content Adjustment After OotimumGene™ Ootimization ............ loo*........ 90 80 Ö 60 c 50 Hi +i 40 c g 30 (j 20 :»-. 10 50 100 150 200 250 300 350 Relative Position of codons 46.72 400 0 50 100 150 200 250 300 350 Relative Position of codons 43.45 400 Average GC content: Figure 2. The ideal percentage range of GC content is between 30-70 %. Peaks of %GC content in a 60 bp window have been removed. Original 1(1) 1(431) 1(394) 1(363) 2(118,371) CIS-Acting Elements Optimized Original E.COli RBS(AGGAGG) 0 0 PolyT( I 1 I I IT) ei 0 Poly A( AAAAAAA) ei 0 Chi sites(GCTGGTGG) [i 0 T7C is( ATCTGTT) [i 0 4. Remove Repeat Sequences After Optimization Max Direct Repeat: None Max Inverted Repeat: None Max Dyad Repeat: None Before Optimization Max Direct Re peat: S i ze: 8 D i star ce: 121 Freque ncy 2 Max Inverted Repeat: None Max Dyad Repeat: None DNA cloning 3. Restriction Enzymes and CIS-Acting Elements Restriction Enzymes Optimized " Green: filtered sites; Blue: checked sites (not filtered); Red: kept sites. NCGl(CCATGG) 1(1) Sall(GTCGAC) 1(431) BamHI(GGATCC) 1(394) Xhol(CTCGAG) 1(363) Hindll I(AAGCTT) 1(371) 5. Optimized Sequence (Optimized Sequence Length:436, GC%:46.72) 6. DNA Alignment (Optimized Region) Conclusion A wide variety of factors regulate and influence gene expression levels, and our Optimum Gene™ algorithm takes into consideration as many of them as possible, producing the single gene that can reach the highest possible level of expression. In this case, the native gene employs tandem rare codons that can reduce the efficiency of translation or even disengage the translational machinery. We changed the codon usage bias in E. coli by upgrading the CAI from 0.61 to 0.74 , and in S. cerevisiae(gbpln) by optimizing the CAI from 0.70 to 0.69 . GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA. The Stem-Loop structures, which impact ribosomal binding and stability of mRNA, were broken. In addition, our optimization process has screened and successfully modified those negative cis-acting sites as listed in the introduction. We are honored to deliver the analysis that you requested. We hope that you are pleased with your GenScript OptimumGene™ results. 4.5. Gene synthesis Comparison of costs □ 1 mutation introduced by QuikChange + recloning ■ working time: 9 hours + 9.5 hours ■ total time: 2-3 weeks + 1 week - price: 7,232 CZK + 2,898 CZK ~ 10,500 CZK □ synthesis of 1000 bp gene + recloning ■ working time: 1 hour + 9.5 hours - total time: 2-5 weeks + 1 week - price: 7,450 CZK h> 16,000 CZK + 2,898 CZK ~ 11,000 -19,000 CZK 4.5. Gene synthesis DNA cloning ÔGenScript The Biology CRO Your Innovation Partner in Drug Discovery! Quotation Radka Dopitova Masaryk University Quote Date: 2011-08-24 Valid Through: 2011-11-24 Quote No. Currency Estimated Turnaround Time Terms Ship Via 985410 United States Dollar {$) Net 30 FedEx Quantity Description Unit Price Unit Discount Extended Price 1 Gene Synthesis: CKIIrd, Len: 436 bp, Vector: pUC57; Cloning site: EcoRV, Quantity: 4 ug S159.00 $0.00 $159.00 Subtotal (United States Dollar) S159.00 $0.00 $159.00 Estimated Shipping handling (United States Dollar] $90.40 Total Quote (United States Dollar) $249.40 Comments: This is only for reference, not an order confirmation. All payments must be made in United States Dollar. This quotation may not include incidental charges, such as shipping and tax. No sales tax is charged for all international orders. The total charge will be determined at the time the order is placed. 40 DNA cloning 4.5. Gene synthesis Model of intermolecular contacts. A B BoxC BoxD CKI1 RD Box A K76 ETR1, RD D CHI_JHD is V -. V v d c eirijhhi id V 1 V M d E CUE! _*ws V d d 9.M lEflSI IDS 113 1 53 v a BehC Pi F ■. Ri : -a 1 is* 1 ] j 1 a V 5 e h V F h.1 Hi f '-C' L . ■ ■ 1 ] L L V a l s e c F. . B a L.>i i | . l a m ~ B h - F e S - x = e S - k z i s -: ft R m s - f. ~ g l B ft fa; us : = i an:' i aH o 41 DNA cloning 4.5. Gene synthesis ai c _o u a CO The Y2H results in comparison to aligmnet of RD domains in AD clones Loop 5 (Box D) L5 - Box D RD AHK1 ETR1 ETR2 EIN4 CKI1 AHK2 AHK3 AHK4 AHK5 d r k ■ m S l d n 1 v l r a m v m l l h n q e e e v l hf e a e q ■ bf e e e n ■ Iv t l q m 42 4.5. Gene synthesis cctggttcagaggaagcaagagaaaccattcaagctggaatggacgcctt: 1 1 1 1 1 1 1 1 1 1 ■ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ■■ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ttagat lAAAGCTTGAATCAAC ttgcaaac 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ■ GTCATT lGAGAAATCGAAA i 1 i i i i 1 i i i i 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 GGACCAAGTCTCCTTCGTTCTCTTTGGTAAGTTCGACCTTACCTGCGGAAC i i i 1 i i &ATCTA 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ľTTTCGAAC TTAGTTGAAC GTTTC i i i 1 i i CAGTAA i j i i i i 1 i i i i j rCTCTTTAGCTTT 700 LVQRKQEKPF KLEWT P S. I k A . I H L U I a L L KSK SWF RGSKRNHSSWNGR LLR KLESTCKRH R N R K Bei I 1 \83rn Hy Psp Not I I Xho I i Sä I Hin dill Eůg I \J JB°3 ľ ! ! I N—^| fisp HI Am/? I CTGGTCATGATCCTGGTTCAGAGGAAGCAAGAGAAACCATTCAAGCTGGAATGGACGCCTTC i i i i I i i i i I i i i i I i i i i I i i i i I i i i i I i i i i I i i i i I i i i i I i i i i I i i i i I i i i i I i i--1 I i i i--1 i i i i I i i i i I i i i i I i i i i I i i i--1 i i i i Xho I :tcgag. Wot dill VAAAGCTTGAATCAACTTGCAAACG G GAT C C Ba m Hl 400 L deletovaná LVMI LVQRKQEKPFKLEWTPSSRK WS SWFRGSKRNHSSWNGRL I N > SGHDPGSEE kodon pro arginin AGA za CGC (zrušení BamHI) Sal I CACTAGGACCCgt cga c Sl^i i ■ I ■ ■ ■ i I i C GAAAT C GAAAGCAAA1 i i i i I i i i i I i i i i I GCTTTAGCTTTCGTTTGCAGTGATCCTGGGcagctg ► r> AKSKANVTRTRR RNRKQTSLGPVD El E S K R H DPS T G I r! kodon pro isoleucirV' ^*Via ATC kodon pro leucin Tn^a CTC • kodon pro valin GTC na GTG • záměna aspartatu (D) za glutamat (E), kodon GAT na kodon GAG • záměna valinu za glycin, kodon GTG na kodon GGG • kodon pro arginin AGA za CGC (zrušení BamHI) Celkový počet mutovaných nukleotidů: 9 Celkový počet mutovaných kodonů: 6 Celkový počet mutovaných aminokyselina- 43 DNA cloning záměna aspartátu (D) za glutamát (E), kodon GAT na kodon GAG záměna valinu za glycin, kodon GTG na kodon GGG Bam Hl GATCC.. . GTCTAGG. BamHI ■GGATCC. .CCTAGG. GATCC. G. 44 DNA cloning Loop exchange cki1_arat l D S l n q l a n v 1 r Old dna tta gat aaa agc ttg aat caa CTT gca aac g t c a t t a g a New dna CTC gag aaa agc ^g aat caa CTT gca aac g G G a T c c g c l E S I n q l a n G I r ahk2_arat p f e e e v l ahk3/2_arat p f e a e q l cre1/2arat p f e e e n l |etri_arat p v S l d n I tc gat aaa c^ gca aac g tl a I I I gaa cgt ttg cj a cj Tl a g l D a n v TT CHAP domain - mutagenesis: cloning strategy atggcgaaaacccaggcggaaattaacaaacgcctggatgcgtatgcgaaaggcaccgtg maktqae inkrldayakgtv gatagcccgtatctcgtgaaaaaaccgaccatctatgatccgcgctttggcgtgatggaa dspy|vkk|t|ydp|fgvme ccgggcgcgattgatgcggatggctattatcatgcgcagtgccaggatctgattaccgat |gaidadgyyhaqcqdlitd tatgtgctgtggctgaccgataacaaagtgcgcccctggggcaacgcgaaagatcagatt yvlwltdnkvr|wgnakdqi aaacagagctatggcaccggctttaaaattcatgaaaacaaaccgagc^^gtgccgaaa kqsygtgfkihenkpsfvpk aaaggctggattgcggtgtttaccagcggcccctatgaacagtggggccatattggcatt kgwiavftsg |i yeqwghigi gtgtatgat^^ggcaacaccagcacctttatcattctggaacagaactggaacggctat vyd|gntstf|ileqnwngy gcgaacaaaaaaccgaccaaacgcgtggataactattatggcctgacccattttattgaa ankkptkrvdnyyglthfie attccggtgaaagcgggcaccaccgtgaaaaaagaaaccgcgaaaaaaagcgcgagcacc ipvkagttvkketakksast ccggcgacccgcccggtgaccggcagctggaaaaaaaaccagtatggcacctggtataaa patrpvtgswkknqygtwyk ccggaaaacgcgacctttgtgaacggcaaccagccgattgtgacccgcattggcagcccg penatfvngnqp ivtri gsp tttctgaacgcgccggtgggcggcaacctgccggcgggcgcgaccattgtgtatgatgaa flnapvggnlpagat ivyde gtgtgcattcaggcgggccatatttggattggctataacgcgtataacggcaaccgcgtg vciqaghiwigynayngnrv tattgcccggtgcgcacctgccagggcgtgccgccgaaccagattccgggcgtggcgtgg ycpvrtcqgvppnqipgvaw ggcgtgtttaaa g v f k Create cloning sites for the cassettes without changing the restriction site. ccatggcgaaaacccaggcggaaattaacaaacgcctggatgcgtatgcgaaaggtaccgtC MAKTQAE INKRLDAYAKGTV gacagcccgtatctcgtgaaaaaaccgaccatctatgatccgcgctttggcgtgatggaa DSPYLVKKPTIYDPRFGVME iccgggcgcgGtCgaCgcggatggctattatcatgcgcagtgccaggatctgattaccgat pga|dadgyy haqcqdlitd tatgtgctgtggctgaccgataacaaagtgAgGccTtggggcaacgcgaaagatcagatt |yvlwltdnkv r p W G N A K D Q I aaacagagctatggcaccggctttaaaattcatgaaaacaaaccgagcttcgtgcctaag |kqsygtgfkihenkps|vpk aaaggctggattgcggtgtttactagtggcccctatgaacagtggggccatattggcatt kgwiavftsgp|yeqwghigi gtgtatgatcccggcaacaccagcacctttatcattctcgagcagaactggaacggctat 'vyd|gntstfiile q n w n g y gcgaacaaaaaaccgaccaaacgcgtggataactattatggcctgacccattttattgaa ANKKPTKRVDNYYGLTHFIE attccggtgaaagcgggcaccaccgtgaaaaaagaaaccgcgaaaaaaagcgcgagcacc IPVKAGTTVKKETAKKSAST ccggcgacccgcccggtgaccggcagctggaaaaaaaaccagtatggcacctggtataaa PATRPVTGSWKKNQYGTWYK ccggaaaacgcgacctttgtgaacggcaaccagccgattgtgacccgcattggcagcccg PENATFVNGNQPIVTRIGSP tttctgaacgcgccggtgggcggcaacctgccggcgggcgcgaccattgtgtatgatgaa FLNAPVGGNLPAGAT IVYDE gtgtgcattcaggcgggccatatttggattggctataacgcgtataacggcaaccgcgtg V C I QAGH I W I GYNAYNGNRV 'tattgcccggtgcgcacctgccagggcgtgccgccgaaccagattccgggcgtggcgtgg Y C P V R T C Q GVPPNQIPGVAW ggcgtgtttaaaTAAGCGGCCGC G V F K Sall-Sall 78 nt Stul-Spel AGGCCT/ACTAGT 117 nt Spel-Xhol ACTAGT/CTCGAG 81 nt Group Restriction sites Red Sail - Sail Red Sail - Sail Red Sail - Sail Red Sail - Sail Red Sail - Sail Blue Stul - Spel Blue Stul - Spel Green Spel - Xhol Green Spel - Xhol Green Spel - Xhol Ncol Kpnl Sail Sail Stul Spel Xhol Natl \\ 1 GTCGAC ......... GTCGAC CAGCTG ......... CAGCTG TCGAC G G CAGCT TCGAC.........A G.........TAGCT GTCGAC ......... ATCGAC CAGCTG ......... TAGCTG Ncol Kpnl Sail xSall Stul Spel Xhol Notl -+