ODODODODODODO □ ODODODODODOD ODODODODODODO DODODODODODOD O D O D O ^ ~ ^ O D O D O D O D ^^1J\^V V D O D ODODOu^uODODO DODODODODODOD ODODODODODODO Kód předmětu: BÍ8980 MASARYKOVA UNIVERZITA Protein expression and purification IV. DNA cloning Lubomír Janda, Blanka Pekarové and Radka Dopitová Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. OP Vzdělávání pro konkurenceschopnost EVROPSKÁ UNIE « NVESTICE I ) ROZVOJE VZDĚLÁVÁNI Název prezentace v zápatí 1 cloning strategy ATGGGCGGCATccACAGGGTGAACAGATGTACCGGAGGGTGTATCGTCTGCATGAGCGCCTGGTAGCCATCCGCACTGAGTACAACCTCC GGCTGAAGGCAGGAGTGGGTGCCCCTGTGACCCAGGTGACCCTGCAGAGTACACAGAGGCGCCCAGAGCTAGAGGACTCCACACTGCGCT ACCTGCAAGACCTGCTGGCCTGGGTAGAGGAGAACCAGCGTCGAATAGACAGTGCTGAGTGGGGCGTGGACTTGCCCAGTGTGGAGGCCC AGCTGGGCAGCCACCGAGGCATGCATCAGTCTATAGAGGAATTTCGGGCCAAGATCGAGCGGGCTCGGAATGATGAGAGCCAGCTCTCCC CTGCCACCCGGGGTGCCTACCGGGACTGCCTAGGTCGCCTAGACCTGCAGTATGCAAAGCTGCTGAACTCCTCCAAGGCCCGCCTCCGGT CCCTGGAG^^^^^^^A ^^^^^^^^l^ririririririAririrpA ririA 7\pp7\pnTn7\nnp^nTP7\ att^ Ar^ a a ana anann_anna an^nn^QCTTTGATT U U, VTVTVTVrpnTVTV^ GGAGTGACC ^ ^ ^ O ^,1 CJ ^5 ^ ^gy1 p ^AAATCAAGG AGATCCAG^ Q. r*1 55 *S 3 H-1 ^ U H 5 H HAGACACAGT GGAGCTGG^ < ^ r< H rj ^ O h rj O H O jTTCGGGAGG O ¥ Hu H Fh Open Reading Frame Stop B. Expression clone sequence: 5' - ACA AGT TTG TAC AAA AAA GCA 3' - [TGT TCA AAC ATG TTT TTT CGT Shiiie-Dalgamo Kozak Open reading frame (amino end) GGC TTC'GAA GGA GAT AGA !ACC? ATG NNN NNN NNN — CCG A|AG GTT CCT CTATCT TGG TAC NNN NNN NNN — ctttBl Translation start* Open reading frame (caiboxy end) --- NNN NNN NNN TAG GAC CCA GCT TTC TTG TAC AAA GTG GT - 3' --- NNN NNN NNN ATC CTG GGT CGA AAG AAC ATG TTT CAC CAI - 51 Translation stop attBl IV. DNA cloning 4.2. The key questions before DNA cloning 4.2.2. Plasmid option 4.2.2.2. Promoters lacUV5, tac and trc promoters are repressed by the lac repressor (lad or lacl«) 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 cI repressor is present. IV. DNA cloning 4.2. The key questions before DNA cloning 4.2.2. Plasmid option 4.2.2.2. Promoters 4.2.2.2.1. T7lac promoter Relative basal uninduced expression levels of cloned p-galactosidase with various vector/host combinations Promoter Host Activity T7 (DE3) 100% T7 (DE3) pLysS 30% T7 (DE3) pLysE 10% T7/oc (DE3) 10% T7/oc (DE3) pLysS 3% T7/oc (DE3) pLysE 1% IV. DNA cloning 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 tac/IPTGor77IPTG Yes pET 77IPTG Yes pGEX tac/IPTG No pBAD araBAD Yes pLEX Pi/trp Yes pPROTet PWanhydrotetracyciin No pTYB T7 IPTG Yes pMAL tac/IPTG Yes pQE T5/IPTG Yes/TOPP pCAL T7/IPTG Yes pFLAG tac/IPTG Biotin binding domain www.promega.com His6, T7 gene 10 www, no va gen.com GST www.amershambiosciences.com His6, GFP www.invitrogen.com Yes His6 www.clontech.com Chitin binding domain www.neb.com Maltose binding domain His6 www.qiagen.com Calmodulin binding www.stratagene.com peptide www.sigmaaldrich.com IV. 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. Arg, Lys, Phe, Leu, Trp and Tyr Tobias et al, 1991, Science Amino acids stabilized in penultimate position N-terminal methionin. His, Gln,Glu, Phe, Met, Lys, Tyr, Trp, Arg Hirel et. al., 1989, PNAS a Lathrop et al. 1992 Liao et.al., 2004, Protein Science IV. 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. Thrombin pH 8.0 PreScission pH 8.9 Factor Xa pH 6.5-7.5 Enterokinase pH 7.0-8.0 Pro-Arg/Gly Pro-Lys/Leu Ala-Arg/Gly Gly-Lys/Ala lle-Arg/Ser Leu-Arg/Ala Ile-Arg/Ile Leu-Glu-Val-Leu-Phe-Gln/Gly-Pro Ile-Glu-Gly-Arg/X Asp-Asp-Asp-Asp-Lys/X AHP2 IV. DNA cloning 4.2. The key questions before DNA cloning 4.2.5. Antibiotic selection ampicillin resistance Ampicillin x Carbenicilin kanamycin resistance IV. DNA cloning 4.2. The key questions before DNA cloning 4.2.6. Codons with translation problems BL21-Codon plus-RIL IV. DNA cloning 4.2. The key questions before DNA cloning 4.2.6. Codons with translation problems http://www.kazusa.or.jp/codon/ 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 . 0 uuc 20 7 ucc 11 2 UAC 13 . 7 UGC 7 2 uua 15.2 UCA 7 . 8 UAA stop UGA stop UUA 12 7 UCA 18 3 UAA stop UGA stop UUG 11.9 UCG 8 . 0 UAG stop UGG 10.7 UUG 20 9 UCG 9 3 UAG stop UGG 12 5 cuu 11 . 9 CCU 8.4 CAU 15 . 8 CGU 21. 1 cuu 24 1 CCU 18 7 CAU 13 . 8 CGU 94 0 cue 10.5 ^■ccc cAc 13 . 1 CGC 26.0 |cuc 16 1 CCCCCC 5 3 CCAACC 185 . 7 CCGGCC 130 8 CUA 5.3^1 CCA 6 . 6 CAA 12 . 1 CGA 4.3 CUA 9 9 CCCCAA 1166 1 CCAAAA 1192 .4 CCGGAA 6 3 CUG 46.9 ccG 26.7 CAG 27.7 CGG 4 . 1 |CUG 9 8 CCCCGG 86 6 CCAAGG 1354 .2 CCGGGG 4 9 AUU 3 0.5 AcU 8 . 0 AAU 21.9 AGU 7 . 2 AUU 21 5 AACCUU 17 5 AAAAUU 2127 .3 AAGGUU 1142 0 |auc 18.2 Acc 22.8 AAC 24 .4 AGC 16.6 AUC 18 5 AACCCC 1108 3 AAAACC 2109 . 9 AAGGCC 1119 3 AUA 3 . l\ AcA 6 . 4 AAA 33.2 AGA 1 . 4 AUA 1 2 6 AACCAA 1155 7 AAAAAA 3204 . 8 AAGGAA 19 0 AUG 24 . 8 AcG 11.5 AAG 12 . 1 AGG 1 . 6 AAUUGG 2242 5 AACCGG 76 7 AAAAGG 3321 . 7 AAGGGG 11 0 GUU 16.8 GcU 10.7 GAU 3 7.9 GGU 21.3 GUU 2171 2 GGCCUU 2188 3 GGAAUU 3261 . 6 GGGGUU 2120 2 GUC 11.7 Gcc 31.6 GAC 2 0.5 GGC 33.4 GGUUCC 1124 8 GGCCCC 10 3 GGAACC 1275 .2 GGGGCC 292 2 GUA 11.5 GcA 21.1 GAA 43.7 GGA 9.2 GGUUAA 97 9 GGCCAA 1175 5 GGAAAA 3249 .3 GGGGAA 24 2 GUG 2 6.4 GcG 3 8.5 GAG 18.4 GGG 8 . 6 GGUUGG 1278 4 GGCCGG 97 0 GGAAGG 3329 .2 GGGGGG 1106 2 Leu-CUA Ile-AUA Pro-CCC Gly-GGA 5.3/7.2/9.9 3.7/7.5/12.6 6.4/19.8/573 9.2/16.5/24.2 Arg-CGA Arg-CGG Arg-AGA Arg-AGG 4.3/6.2/6.3 4.1/11.4/4.9 1.4/l2.2/19.0 1.6/12.0/11.0 IV. DNA cloning 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 30CC). • Free energy of unfolding is observed to be only 5-20 kcal/mol. • Consequently, proteins are on the verge of denaturation. IV. DNA cloning 4.3. Protein solubility 4.3.1. Determining surface charge vi Solubility af p - laetoglobulbi at various fNaCI] 1 2.0 i1 1.0 .001 M 5.0 5.2 5.4 5.6 pH Most precipitation J~ > so, I.E.P. pH po*- > sol- > coo" > cr NIIÍ > 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. IV. DNA cloning http://www.roselabjhu.edu/~raj/MISC/hphobh.html Heme Binding? cytosol Proline Rich Predicted transjKAbrane alpha helixes jdl-19% IV. DNA cloning 4.3. Protein solubility a o o o i . . http://www.biotech.ou.edu/ 4.3.3. Solubility model The revised Wilkinson-Harrison solubility model CV -X^T") + X21 (T-'- 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 11,12 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')2 IV. 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, Gln, Asn) were on the surfaces of the proteins studied. • The interiors were primarily composed of aliphatics (Gly, Ala, Ile, 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 1 13 Leu L 11.7 41% Val V 10.9 Cys C 8.4 47% Trp W 7.9 23% Ala 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% Gln Q -17.1 6% Asn N -20.1 10% Glu E -34.3 20% Lys K -36.8 4% Asp D -38.5 15% Arg R -51.4 0% IV. DNA cloning 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-NO2 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% 0.75 Ala A 6.7 38% 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 Gln 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% 1.04 IV. 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 V L N E N L L R F F V A in a-casein makes the molecule more soluble. •Phenylalanine residues are likely to self-Interact and are frequently found at subunit interfaces. 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% 0.75 Ala A 6.7 38% 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 Gln 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% 1.04 IV. 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, andArg. • 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 Ile over Leu, Ala and Met. • Most of these changes occur in a-helical regions and increase the net hydrophobicity of the residue. Amino acid Transfer free energy Chou-Fasman kJ/mol % buried coil index | Phe F 15.5 48% 0.71 Met M 14.2 0.58 | Ile I 13 IBS 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% 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 Gln 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% 1.04 IV. 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 0.58 lie I 13 1SI 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% 0.7S Ala A 6,7 38% 0.7 Thr T S 2S% 1.07 Gly G 4,2 37% 1.S Ser S 2,S 24% 1.82 Pro P -0,8 24% 1.S9 Tyr Y -2,9 13% 1.06 His H -12,S 19% 1.06 Gln Q -17,1 6% 0.86 Asn N -20,1 10% 1.3S Glu E -34,3 20% 1.2 Lys K -36,8 4% 0.98 Asp D -38,S 1S% 1.2 Arg R -S1,4 0% 1.04 I. The molecular p :eins - Lubomír Janda Please solve the problem. Question 1: I am a promoter. I am present on the DE3 lysogenic phage. 5 points In expression strains, I am very often found before the T7 RNA polymerase gene. 3 points A lac operator is present in my sequence. 2 points Originally, I am found before a gene encoding lactose utilization protein. 1 point lac promoter 1. The molecular principles for understanding proteins - Please solve the problem. Question 2: 1 am an amino acid. 1 am a positively charged amino acid with absence of C - e. 5 points Expression of my tRNA is reinforced in E. coli strain BL21-Codon plus-RIL. 3 points In terms of structure, I am not a burried amino acid. 2 points. In one letter coding, I am designated R. 1 point Arginin IV. DNA cloning 4.4. Gene cloning 4.4.1. Gateway cloning for protein expression PCR Product attB1 attB2 GOt BPQonase select for Spc resistance 3ttR1 attR2 Destination Vector LR Gonase select for Amp resistance Expression Clone GO! = gene of interest SpcR = spectinomycin resistance gene Ampfl ~ ampicillin resistance gene prom = transcriptional promoter ccdB = toxic negative selection marker CAT = chloramphenicol resistance gene IV. DNA cloning 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 //— NNN //— NNN ACA AGT TTĚ TAC AAA AAA GCA GGC TNN TGT TCA A AC AH TTTI TTT CGT CCG AÍN H From affFM From Destination ~~ Vector :GENE^ From aftt_1 From Entry_ Clone /AGEHE- _ NNN NAC CCA GCT T Y K V V TTG TAC AAA GTG GT|N NNN _// NNN NÍTG GGT CGA AAG AAC ATG From atfL2 From Entry Clone TTT CAC CAN NNN From attR2 From Destination Vector : IV. DNA cloning 4.4. Gene cloning 4.4.1. Gateway cloning for protein expression GOI-stop GOI-nonstop Kozak-GOI-stop TEV-GOI-stop Aminoterminal fusions Aminoterminal and/or carboxyterminal fusions Aminoterminal fusions or native eukaryotic expression 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 IV. DNA cloning 4.4. Gene cloning 4.4.2. Flexi vector cloning http://plasmid.hms.harvard.edu Ligation-dependent cloning method facilitated by selection for the replacement of a toxic gene insert in an acceptor vector. Cloning efficiency: Human Mouse Rat C. eIegans Zebra fish Arabidopsis Yeast 98.9% 98.9% 98.8% 98.5% 97.8% 97.6% 97% NcoiO 14) Pact (1254) S^fl (1320) 1200 1400 mu ľ fiÖÖ 1600 11000 h 200 11400 MáOO ! Hl»8-MBP-TeV- 1 ^> I Bwn*M ~~^> [ 1800 2000 12200 CAT "T24OO Pm&\ (2452) Hrdlil (2665) MBP Forward sequencing primer I "1^00 [2SÖÖ Í3OO0 "^3200 Í34ÔÔ Í3600 ľäŠÔČ [4ÖÖÖ I«oÖ TwOO [4600 [«OO Uctg 77 Terminator Reverse sequencing primer 3f sequence homofogy region AvrW (6164) ßs/WI (7133) "Tiööo fiäöö [540Ö Í6Š00 ÍŠ8QO ieööö leiöö í&íoo leeoo IsaücT" ITööö" 7200 pBR322_qľi9in Kan R&al&tancg B I GAAAACCTGTACTTCCAGgcga tcgcGGCC + + + c pme\ I GGGGCGTAATgtttaaacGAATTCGAGCTC > i I i i + + + CTTTTGGACATGAAGGTCcqctagcgCCGG I E N L Y F Q A j I A A CCCCGCATTAcaaatttgCTTAAGCTCGAG G A slon IV. DNA cloning gene-specific primer A I ASVDPACP GGTTgcgatcgcCAGTGTGGATCCAGCTTGTCCC....... I i i i i | i i i i | i i i i | i i i i I i * TCACACCTAGGTCGAACAGGGGTTTCGA > N-terminus of target V D C-terminus of target > MNMQPEDV stop AT GAACAT GCAAC CT GAAGAC GT GT GA............... I M I | I I I I | I I I I j I I I I | I I I I | I I I I | I 1 I I | I I I I | 1 I .....ACGTTGGACTTCTGCACACTATCcaaatttgTGTG gene specific primer I Pmel addition I Q P E D V stop IV. DNA cloning 4.4. Gene cloning 4.4.2. Flexi vector cloning fiac\ (1254) NCO\0 14) Sgf\ (M02) j 1200 400 600 IbOO íiooo 11Í-00 i1400 11600 hsoo i2000 S3 C HkS-MSP- I > I Olm»» PtPe\ (2434} M£P Forward primer HinölW £2647) 2200 12400 12600 12800 I 3000 32O0 3400 13800 14O0O Í4200 CAT 3 C 77 Terminator Reverse primer AvrW (5264) fisiWI(6233) 14400 4ÜÜ0 Í4&00 5000 Í5200 J540O fseoo Í58O0 leooo 16200 j>BR322 Origin I Kan Cassette QMt gcgatcqccGAAAACCTGTACTTCCAGTCCGTGGATCCAGC i M t > i t > f i t i i I i 1 i i t i t i i l i 1 i i I t i i H i"' ' ■ I ' ■ ) ' i CrTTTGCSAt^TG/vACKŤTCAGGCRfJCTAíiCíTCG 1st PCR; gare-specinc * 3 TEV primer~J> flrTWl ATGCAACCTGAAGACGTGTGA Sud PCR: 6F Sflfl + F TEV primer TACGTTGGACTTCTGCACACTa tcaaa t11get taagetegagtqtg <^ í"ň?Cfi:" fl*ne»peeffle + Fmei *w l 2nd PCR: ttcv*r** primer J IV. DNA cloning 4.4. Gene cloning 4.4.3. In-fusion PCR cloning http://bioinfo.clontech.com/infusion/ 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. AmpR \^ Lef-2,603 Origin (PUC) CM V enhancer Chicken actin promoter Orf1629 T7 terminator beta globin poly A signal T7 promoter/lac operator p10 promoter + 5' UTR Infusion site ( Kpnl) N-HIS-3C tag lacZ promoter/gene insert 3' infusion site (Hlndlll) IV. DNA cloning 4.4. Gene cloning 4.4.3. In-fusion PCR cloning Kpnlj 5' OTGGAAGTTCTGTTTCAGGGTAC 3' GACCTTCAAGACAAAGTCC L E V L F Q G Partial 3C protease site Hindlll | Linearized vector (pOPINF) AGCTTTCTAGACCAT 3' AGATCTGGTA 5' AAGTTCTGTTTCAGGCCCG TTCAAGACAAAGTCCGGGC Gene/domain of interest TAAAGCTTTCTAGACCAT ATTTCGAAAGATCTGGTA Stop In-Fusion ready PCR product In-Fusion enzyme 42 C 30 minutes 5' CTGGAAGTTCTGTTTCAGGCCCG 3' TTCAAGACAAAGTCCGGGCCGGC LE VL F Q ▼ G P Full 3C protease site Gene/domain of interest Hindlll TAAAGCTTTCTAGACCAT ATTTCGAAAGATCTGGTA Stop Expression/transformation ready construct IV. DNA cloning Vector Fusion tag Pa renl vector/a ntibioti c Restriction sites lor lin-r rj s i a la nee e ari za ti on of the vector Forward primer extension Approximate increase in size of PGR product Reverse primer extension wfthT7 primer (bp) pOPINA ...KHHHHHHug pET2 8a/Kanamycin Ncol and Dral AGGAGATATACC&IG GTtiGTGGTGGT-GTTT 110 pOPINB MGSSH H H HH HSSGLEVL-FQOGP „, tag p ET28 a/Ka* lamyc i 11 Kpnl and Hindlll AAGTTCTGTTTCAG-GGCCCG* ATGGTCTA* GAAAGcrrri^ 130 pOPINC .„KHHHHHH tag pTriEx4/Ampidlli n Ncol and Pmel AGGAGATATACCATGt GTGATGGTGAT-GTTP 200 MAHHHHHHSSGLEVL-FQOGP... tog pTriEx4/Ampidllin Kpnl and Hindlll AAGTTCrGTTTC AG -GGCCCG* ATGGTCTA-GAAAGC^Häi 225 pOPINE .„KHHHHHH tag pTri Ex2/Ampicillin Ncol and Pmcl AGGAGATATACCdXQ^ GTGATGGTGAT-GTTV 170 pOPINF M A f! H H H H H SSGI fA' L -FQOGP . tag pTriKx2/AfTipic iJ tin Kpnl and Hindlll AAGTTCTGTTTCAG-GGCCCG* ATGGTCTA-GAAAGCTHAj 225 pOPFNG MGILPSPGMFALLSLVSLLSVLL pTriEx2/AmpLdUin MGCVAOETG... deavabk Kpnl and Pmel GCGTAGCTGAAAC;CGGC GTGATGGTGAT-GTTT 260 secretion leader and .KHHHHHH lags pOPINH MGILPSPGM PALLSLVSLLSVLL MGCVAUETMAHHHHHHS SGLEVLFQUGP dcavablc secretion leader and dcavablc N-his tag pTriEx2/Ampiälli n Kpnl and Hindlll AAGTTCTGTTTC AG -GGCCCG1 ATGGTCTA* GAAAGCTTX4 315 pOPINI MAHHHHHHSSC.tag pTri Ex2/Ampic illin Kpnl and Hindlll ACC ATC ACAGCAGCGGC ATGGTCTA-GAAAGCTHa 200 pOPINJ MAH H H HHHSSG GS1 ■ LEVLFQOGP, >ug pTri Hx 2/Ampu i lun Kpnl and Hindlll AAGJT< U.ri'K AG i. it i( i < G* A1U.K IA GAAAGY") STOP-cgcgac-Pac 1 j Kan r Deletions (ALMN): Primer 7 -> Protein of interest sequence: Primer 6 Insertions (M INS <- N): Primer 9-> Protein of interest sequence: Primer 10-> TCAG CGA TCA ACA GTA TGG TAG 3' ACDEFG HIKL MNPQRSTVWY 3'-ACG CTA CTT AAG CCC GTG TAT Ijr;^ 0 j** CCG CAG 0A TCA ACA GTA TGG TAG' DEFG HIKL M NP Q R S T V W Y ♦- 3-ACG CTA CTT AAG CCC GTG TAT TTC Gn^^ XAAC OCT CAG CGA TCA ACA GTA TGG TAG' ACDEFG H I K LM NPQ R STVWY — 3-ACG CTA CTT AAG CCC GTG TAT TTC CCT IASJto rr* IV. DNA cloning T7 terminator _1_ Hmdlll Bamlll IV. DNA cloning -CTGTACTTCCAATCCAAT -GACATGAAGGTTAGGTTA T4 polymerase CTG GACATGAAGGTTAGGTTA ATTGGAAGTGGATAACGG TAACCTTCACCTATTGCC dGTP ATTGGAAGTGGATAACGG GCC- TACTTCCAATCCAATGCX ■ ATGAAGGTTAGGTTACGY ■ --TAACATTGGAAGTGGATAA --ATTGTAAC CTTCACCTATT T4 polymerase dCTP TACTTCCAATCCAATGCX----TAAC CGY----ATTGTAACCTTCACCTATT Annealed (N-terminal side) LYFQSNA------ - - -CTGTACTTCCAATCCAATGCX- - -........------ - - -GACATGAAGGTTAGGTTACGY...........------ IV. DNA cloning 4.4. Gene cloning 4.4.5. LIC vectors Vector Parental vector Antibiotic Leader sequence MW (leader)9 kb Purpose PMCSG7 pETJla* Amp N-His-TEV-LIC 2,755 5,286 production pMCSGS pMCSG7 Amp N-His-Sloop-TEV-LIC 4,399 5,341 toxicity PMCSG9 $MC$G? Amp N-His-MBP-TEV-LIC 43,713 0,147 solubility pMCSGlO pMCSG7 Amp N His-GST-TEV^ LIC 29,046 5,961 solubility pAlCSGl 1 pACYO Cam N-His-TEV-LlC V55 4,071 coexprc&sion pMCSG12 pACYC-Duet-1 Cam N-His-Stoop-TEV^ uc 4,399 4,144 compression pMC$Gl3 pACYC-Duct 1 Cam N-His-MJlP-TEV-LIC 43,713 ■^940 compression pMCSGH pACYC-Duei-1 Cam N-His-GST-TEV-LIC 29,046 4,754 compression pMCSG17 pMCSÜ7 Amp N-Stag-TEV-LIC 3,760 5,316 coexprtssiort pMCSG19 pMCSG? Amp N-MBP-TVMV-His-TEV-UC 45,050/ 2,711" 6,441 production pMCSG20 pMCSGl? Amp N-Scag^GsST-TEV-UC 30,051 5,991 coexpression pMCSG21 pCDFDucť lc Spec N-His-TEV-LIC 2,755 3,852 coexpression pMCSG22 pCDF- Spec N-His-Sloop-TEV- 4,390 3,906 coexpression pMCSG23 pCDF-DueM Spec N-His-MBP-TKV-LIC 43,713 4,971 coexprcssiou pMCSG24 pCDf-Duet-1 Spec N-His-GST-TEV-LIC 29,046 4,527 coexpression IV. DNA cloning 4.4. Gene cloning 4.4.6. High-throughput cloning and protein expression analysis Process Workflow Stage 1: Vector annealing and cell transformation (Prepared with Robots) Stage 2; Plating for individual done selection (Prepared Manually) Stage 3: Overnight growth @37°C Stage 4; Transfer select colonies into Bacterial growth cultures Stage 5: Remove aliquot as a temporary freezer stock 96 wefi plate of Transformed cetis \ 4B mil agar done selection plates l™1-i- G i 4$ Deepwell plates of Bacterial growth cuitures • 1 48 De#pweH 0 48Dwpv*Hl 14 \ / \ 96 weil plates of temporary freezer stocks IV. DNA cloning 4.4. Gene cloning 4.4.6. High-throughput cloning and protein expression analysis Stage 6: IPT6 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 7. 8. Tep 6. #1 #2 4SD*«pweti B \ / \ 96 well plates ofprotein expression samples i i i © i i I 3 Process aif four 4B Deep well plates of Bacteria! growth cultures for solubility screening and process aft two 9G weft plates for protein expression screening I. The molecular principles for understanding proteins - Lubomír Janda Please solve the problem. Question 3: To which cloning strategy does this issue belong? The ccdB gene encoding protein which binds DNA gyrase. 5 points This protein does not interact with small molecules. 3 points It is a motor protein. ^^^^^^^^^1 2 points This protein interacts with microtubules. 1 point Third protein group (macromolecule-binding proteins) I. The molecular principles for understa Please solve the problem. Question 4: What is the name of this posttranslational modification? I modify arginine, lysine and five other amino acids. 5 points I am detectable by MALDI and western blot (but not for all kinds of modified amino acids). 3 points Very often I need ATP for the modification. 2 points Serine and threonine are the most used amino acids for this modification. 1 point Phosphorylation