onononononono nonononononon OnODODOnODODO □ onononononon o □ o □ o " ~ " o d o □ o JJIj Kód předmětu: C8980 I UfU * MASARYKOVA UNIVERZITA ODODOu^LiODODO □onononononon onoDonoDODOno Protein expression and purification • IV. DNA cloning Lubomír Janda, Jozef Hritz, Blanka Pekárovi, Radka Dopitová and Adam Norek Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. HHf £5 cl) ^í^^fc I MINISTERSTVO ŠKOLSTVÍ, OP Vzdělávání EVROPSKÁ UNIE ^■^r ■ pro konkurenceschopnost ^WA^ 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,-Ar,r,r ej h < 0 b 1 u u b 0 i I I—I 1 0 "JO O o "~> >r. OS o-\ 1 I M I_, 41 'i\r,r,i\i\r,r,i\rT,Tr,i\' U ,r,rrr,r,r,rrr,-A-Arrr,-Ar,-A-A-Ar,-A-Ar,-Ar,r,-Ar,r,-A-Ar,r u b b S-i ■< u se r.1 i-H II 2 S ± 5 d □ 6 q «3 ^ < h 5 w o 4 1 . »-h £? 00 ^ f-M >—i ■< &5 cq h u o I 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 http://www.compbio.dundee.ac.uk/jpred/ jpred4 mastdsesetrvksvrtgrkpignpedeqetskpsddeflrgkrvlvvddnfisrkvatg —eeeeee----eeeeeeeeee-----------------.-^-'--eeeeee—hhhhhhhhh KLKKMGVSEVEQCDSGKEALRLVTEGLTQREEpG^vdklpfdyifmdcqmpemdgyeatr hhhh----eeeee—hhhhhhhhhh-------------eeeeee------hhhhhh eirkveksygvrtpiiavsghd^@seearetiqagmdafldkslnqlanv.jreieskrh hhhh---------eeeee^--—hhhhhhhhhh-----e---HHfJHtfTHHHHHHHH--- .................. 4.2.1.3...Dt5mains detected by SMART ......... httg^fnart.embl-heidelberg.de/........S1\7lART ^rvlvvddnfisrkvatgk^kkMgvseveqcdsgkealrlvtegltqreeqgsvdklp fdyifmdcqmpemdg.¥-e^treirkveksygvrtpiiavsghdpgseearetiqagmda FLDKSLNQLANVl"* Confidently predicted domains, repeats, motifs and features: Name Begin End E-value REC 43 171 1.19e26 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. 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% 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 771 PTG Yes pET 771PTG Yes pGEX toc/IPTG No pBAD oro BAD Yes pLEX Pí/trp Yes pPROTet P/.řeř/a n hyd rote t ra eye 1 i n No pTYB 771 PTG Yes pMAL toc/IPTG Yes pQE 75/1PTG Yes/TOPP pCAL 77/1PTG Yes pFLAG 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 10 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 ii 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 13 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 16 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. 18 DNA cloning 4.3. Protein solubility 4.3.2. Determining hydrophobicity http://www.roselabjhu.edu/~raj/MISC/hphobh.html 19 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')220 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 Leu L 11.7 41% 1 Val V 10.9 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%] 21 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 50% 0.58 lie 1 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% ■■ 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% 1.Ó* 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 acic I 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% 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% i32 Arg -51.4 0% 1.04 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 I Met M 14.2 50% 1 0.58 lie I 13 65% J 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% 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% im 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 acic 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% 1 0.62 Cys C 8,4 47% 1.18 Trp W 7,9 23% j 0.75 Ala A 6,7 38% 1 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% 1.026 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 ma^r 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 TAG AAA AAA GCA GGC TNN TGT TCA A AC 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 Tit 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 27 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 MGSDKIHHHHHHENLYFQG CCCiB STOP-cgcgae-Pac 1 SpeedET Primer 1 Kan r 29 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 TEV si ± Insert . His tag lac operator \^ T7 promoter I RBS \_ e LIC site Ssp Leader Sequence Nde I (ATG) Bglll Kpnl Hzndlll BamlU T7 terminator 30 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.....------------ 31 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 5 96 well plate of Transformed cells 48 well agar Clone selection plates —I—I r T" I I I * L T«0 1 O i 48 Oeepwell plates of Bacterial growth cultures / \ / \ 48D««pweorgti= expressJar. Inclining tut not imibed ;s: Codon ui-*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 34 Results E coll 1. Codon usage bias adjustment 2. GC Content Adjustment 3. Restriction Enzymes and CIS-Acting Elements Restriction Enzymes Optimized Original * Green; rBtoed -jrces: h.'le-: jftrriteir :r»r ftt=r= □ □ Wcol{CCATGG) l(1j Ndel(CATATG) u 0 NotUGCGGCCGC'i 1(2408) 1(24GS) Fsll(CTGCAG> D 0 Pvul(CGATCG) D =:■ S3Gr(GAGCTC) D □ Sall(GTCGAC} 0 SmaUCCCGGG) □ : Sphl(GCATGC) D □ Stu-I(AGGCCT) D d XhoKCTCGAG) ■:■ 0 BgHI(AGATCT) D 1f737) XbalfTCTAGA) 0 1(947) BctlfTGATCA) 0 2<18t4.20DDj Polymerase sli ppage site 1 0 0 Polym erase slippage site 2 D 0 Frameshift element P 1(420) Rjbc-same binding site D □ 5. Optimized Sequence (Optimized Sequen ce Length 528, GC%:56.12) 6. DNA Alignment (Optimized Region) GMACCCAAA32JUL7GCCCACTCCCTC<3aGCGETGCT ~TCTGÍJTGňT\3CCG^TG □ TG^TGATGCC^CT^OÍIňTTATTGeGCflGG^ ["TAAC CAAOGTAG CAG C A wide variety of factors regulate and influence gene expression levels, and our OptimumGeneIM 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 D.35 to 0.93 . GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA. The Stem-Loop structures, which impact ribosonial 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 QptimumGene™ results. optimized s GAAACCC °r±ginal 5 GCGAT"TA optimized ce Conclusion 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 DNA cloning Radka Dopitova Masaryk University Quote No. 985410 Gene Synt Quantity: 4 Comments: This is only for reference include incidental charge determined at the time tt Quotation 1275USD/6000bp=0,21 USD/bp Lutujrr-:' Jsnda Quale Data: 301 $-0 ťele' nery Resaarcn InsiitulB valid Through: S í) I Quota No. Currency Essmated Business. Day Terms, B^lp Via. Un-led Staieiv lar 17-22 Nat 30 DHL lnem ko Ouanuly Dsacnpiian UrutPnce i -; Disc. Esaandeo Price 1 Gene 5ynrfteais:?(PCAD Len: 1. ■. 1 5bp, Vector nama: pUG57, Plasmid pfepatatsm: Standard delivery: 4 ug (Free cd crtarge} (shippableí Ii. $fl9S.90 3 1 Ge.ne £yntňeais:AmiS Len: l,£45fcp, Vector na™: pläCS7, Plasmid pjepa/alian: Slanoara delivery: it pg iFnee of charge) (shippahle} S23e_35 $33.(4 S3Ě7.71 3 1 Ůene SyntnesiB:CHiF Len: t ,313fcp, Vector name: pUC57_ Plasmid pneparalien: Slandarc delivery: t pg (Free of :harge'i (shippable} W1&.7Ě $41.«$ S376.0B 4 1 Gene SyrrtnesiE,:£)PLL-3i Lan:52abp, Veüorname: pUC57. Plasmid preparation: Slandard delivery: i pg (Free -'. zr.arqí' jihippatriej S 131.44 $12. E4 Sics.ao SubtotaljUniied States DollarJ i.aäťt.on t 133.01 $ i.3JI í)3 Eíf:maied S*ipp;ng"Handling.:-Jnir-ed S lb tet. Dollar) $33. jy Printing Fee | Untied Sla!as Doiiar;- JO.D0 Total Quote (Esatľlufljng VAT} S 1,375.3a Promotion code Gustorrized Disc □um PrcmoLon coda expired day Incc-Terms K;ndry note: International clients are responteiie frj pay duly and tax 1or cusCotTe Clearance purpose, tf your organrzaöon has duty or tax exemption eertriL=_ie. please sena it Iď ourlechnical account managers Quote Date: 2011-08-24 Valid Through: 2011-11-24 Terms Met 30 Ship Via FedEx Unit Discount S0.00 $0.00 tates Dollar) tates Dollar) Extended Price $159.00 $159.00 $90.40 $249.40 This quotation may not The total charge will be 38 DNA cloning 4.5. Gene synthesis Model of intermolecular contacts. A B BoxC CKI1 RD Box A BoxD K76 ETR1, RD D chi_jhd is V -. V V D C EiRijnn id V 1 V m D e cue! _*ws V D D 9.h9 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 39 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 40 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 Hl Am/? I CTGGTCATGATCCTGGTTCAGAGGAAGCAAGAGAAACCATTCAAGCTGGAATGGACGCCTTC CTCGA 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 i i 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 GACCAGTACTAGGACCAAGTCTCCTTCGTTCTCTTTGGTAAGTTCGACCTTACCTGCGGAAG GAGCTCTTTTCGAACTTAGTTGAACGTTTGd Xho I Hin dill G \AAAGCTTGAATCAACTTGCAAACd Bam HI I G GAT C CJG i I i i i i CCTAGG C 400 L deletovaná LVMI LVQRKQEKPFKLEWTPSSRK WS SWFRGSKRNHSSWNGRL I N > SGHDPGSEE kodon pro arginin AGA za CGC (zrušení BamHI) Sal I CACTAGGACCCgt ega 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' V*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- 41 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. 42 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 ttg 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 ctt gca aac g tl a i i i gaa cgt ttg cj a cj Tl a g l D a n v TT >spI Q15149-7 Iplec_human Isoform 7 of Plectin OS=Homo sapiens gn=plec MKiJderdrvqkktftkwvnkhlikaqrhisdlyedlrdghnlisllevlsgdslprekgrmrfhklqnvqialdylr hrqvklvnirnddiadgnpkltlgliwtiilhfqisdiqvsgqsedmtakeklllwsqrmvegyqglrcdnftsswrdg rlfnaiihrhkpllidmnkvyrqtnlenldqafsvaerdlgvtrlldpedvdvpqpdeksiityvsslydamprvpdvq dgvranelq Ncol ccatggjagatcgtgcccgatgagcgggatcgtgtgcagaagaaaaccttcaccaagtgggtc ME IVPDERDRVQKKTFTKWV aacaagcacctcattaaggcccaacgtcacatcagtgacctgtatgaagacctccgcgat NKHLIKAQRHISDLYEDLRD ggccacaacctcatctccctgctggaggtcctctcgggggacagcctgccccgggagaag GHNLISLLEVLSGDSLPREK gggaggatgcgtttccacaagctgcagaatgtccagattgccctggactacctccggcac GRMRFHKLQNVQIALDYLRH cgccaggtgaagctggtgaacatcaggaatgatgacatcgctgacggcaaccccaagctg RQVKLVNIRNDD IADGNPKL EcoRV acccttggcctcatctggacaatcattctgcacttccagatctca|gatatc|caggtgagt TLGLIWTI ILHFQISDIQVS gggcagtcggaggacatgacggccaaggagaagctgctgctgtggtcgcagcgaatggtg GQSEDMTAKEKLLLWSQRMV gaggggtaccagggcctgcgatgcgacaacttcacctccagctggagagacggccgcctc EGYQGLRCDNFTSSWRDGRL ttcaatgccatcatccaccggcacaagcccctgctcatcgacatgaacaaggtgtaccgg F N A I I HRHKP LL I DMNKVYR cagaccaacctggagaacctggaccaggccttctctgtggcggagcgggacctgggagtg QTNLENLDQAFSVAERDLGV acgcggctcctggaccctgaggacgtggatgtccctcagcccgacgagaagtccatcatc TRLLDPEDVDVPQPDEKS I I acctacgtctcgtcgctgtatgacgccatgccccgcgtgccggacgtgcaggatggggtg TYVSSLYDAMPRVPDVQDGV Xhol Stul BamHI tga -k agggccaac RAN CTCGAG AGGCCT GGATCC R 44 Ncol ccatggagatcgtgcccgatgagcgggatcgtgtgcagaagaaaaccttcaccaagtgggtc MEIVPDERDRVQKKTFTKWV aacaagcacctcattaaggcccaacgtcacatcagtgacctgtatgaagacctccgcgat N K H LL I KAQRHI S DIiYE DIiRD ggccacaacctcatctccctgctggaggtcctctcgggggacagcctgccccgggagaag GHNIiISLIiEVIiSGDSLPREK gggaggatgcgtttccacaagctgcagaatgtccagattgccctggactacctccggcac GRMRFKKIiQNVQIAI.DYI.RH cgccaggtgaagctggtgaacatcaggaatgatgacatcgctgacggcaaccccaagctg RQVKLVNIRNDDIADGNPKL EcoRV T7 promoter primer #69348-3 T7 promoter _lac operator_ xpal lbs PET upstream primer #69214-3 . JSjfll AGATCTCGATCCCGCGAAATTAATACCACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAACAAGGAGA Wco i His-Tag /Wei Wriel T7-Taq_ TATACCATGGGCAGCAGCCATCATCATĽATCATtACAGLAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAC; CATGACTGGTGGACAGCAA MclGlySo-So-MuHl sH isHlsH i sH I sSg-5o-G I yl ovYci I P-oA ■ qC I ySo - H i s Ho IA J oSo-Not Th -C I yC I yG I nC I r Sa/T Hind III Noil Xho\ BsfrU \EcoR Sac I thrombin His-Tag ATGGGTCGCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pl~T 28o ( '! HstGIyA-gClyS.-CIuPheCIuLfluA-gA-gGI r>AIoCylGIyA-gTb-A-gAIoP-oP-oP-oP-oP-oL.uA-gSe-CIyCylEnd .GGTCGCGATCCGAATTCCAGCTCCCTCGACAACCTTGCCGCCGCACTCCACCACCACCACCACCACCACTGACAKCGGCTGCTAACAAAGCCC pET-28b(•! - G IyArgAspP-oAsnSe-So-Se-ValAipl ytl euAIaAIaAIol auGIuHIiHIiHI»HllHI«HI fEnd ..GGTCGGATCCGAATTCGAGCTCCGTCCACAAGCTTGCGCCCGCACTCGAGCAC CACCACCAC CACCACTGAGATCCCCCTGCTAACAAAGCCC pET-28c(■) ..GlyA-qlleA-gl I e H-qA I nP-pSo-Th-So-l.au A-qP-pH i sSe-So^Th-Th-Th - Th-Th-Tb-G lull nA-qlnul ouTb-[. ySP-o_ of ;;1 ■ 32 T7 terminator GAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG T7 terminator pnmer #69337-3 pET-28a-c(+) cloning/expression region ------3----3 — 3 — ---3 — 3----a — TYVS SI.YDAMP Xhol Stul BainHI 3 ~ --3 3--3 ~ 3--3 3--3 ^ ^ 3 ~3 R V D V Ľ V agggccaac[CTCGAGJ |AG GC C T| t g a|GG AT C C RAN L E R P w E S 1. Ncol-Xhol do pETM60 2. Ncol-Xhol do pET28 3. Ncol-BamHI do pETM60 4. Ncol-BamHI do pET28 K expresi používáme štandartne dva vektory: pET28 pETM60Ubq Dra IIK5127) X no 111») Nat ii Eag K1661 Hind 111(173) Sal 1(1791 Sac I[1301 ECOR 1(1921 BamH K1S6] N he l{231) N de II238) N co 1(296) XDa 1(335) Bgi linou SgrA l(+«2) Sph H59B) \,MIU K1123I IV NBCI 1(1137) "j j SBStE IKI304! V3 Apa n 7 7BSSH 11(1534) /ECOR V(1573) > Hpa 1(1629) bsss 1(3397) BspLU11 K322ÍÍ Sap 1(3108) Bstl I07 1(2995) Tth 111 1(2969) PShA 1(1968) Bgl 1(2197) Fsp 1(2205) PspS 11(2230) Ubq-His.tag-TEV-ABD-His.tag-STOP ABD-His.tag-STOP Ubq-His.tag-TEV-ABD-STOP ABD-STOP 45