1

W1_ DesignYourExpressionStrategy The goal of this workshop is to establish a workflow which allows to make a rational decision upon gene design, regulation of gene expression, protein targeting, vector system and platform strain for the design of a suitable expression strain. This decision is also influenced by: + + + + + Possible advantages for expression by co-expression of other proteins such as chaperons The available cultivation system and DSP equipment The application of the produced protein as isolated protein or in whole cells The desired quality of the protein and many other factors.

Many successful strategies for the design of optimized expression strains start with detailed analyses of the target sequence. The following links provide access to servers for simple sequence analyses which allow to predict important properties of the target protein and should influence the design of a suitable expression strategy and constructs before a decision is made about the use of: + + + + + a synthetic or native gene a suitable vector/promoter a multi copy/low copy strategy an exchanged signal/targeting sequence an optional fusion with another protein or tag

 Start with protein sequence analysis: + + +

Clarify origin (native host): blastx, blastp http://blast.ncbi.nlm.nih.gov/ Homologs in Pichia? blastx, blastp http://blast.ncbi.nlm.nih.gov/ Analyze physical/chemical data: Mw&PI:http://web.expasy.org/compute_pi/ AA comp&PI: http://web.expasy.org/protparam/

Analyze targeting/localization: Psort: http://psort.hgc.jp SignalP: http://www.cbs.dtu.dk/services/SignalP/ http://www.scilifelab.se/archive/pdf/tmp/signalp_paper.pdf TargetP: http://www.cbs.dtu.dk/services/TargetP/

+ + + +

Analyze posttranslational modification : http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp N-glyco, phosphorylation, cofactors etc http://prosite.expasy.org/ Cofactor binding: http://pfam.sanger.ac.uk/search Disulfide bonds: http://disulfind.dsi.unifi.it/process.php ..

Metaserver: Predictprotein: recommended but needs registration http://www.predictprotein.org/

Analyze literature about homologous proteins expressed in Pichia and other hosts

Each group should decide for one example sequence, analyse this sequence and then suggest a suitable expression strategy. For example: A protein is predicted to be secreted in the native host, the native host is a plant, the protein contains disulfide bridges and is N-glycosylated. High yields of cheap protein are desired for biocatalytic applications where small protein modifications or heterogeneities do not matter. Consequence: Design synthetic gene for expression as secreted protein in Pichia pastoris, use strong methanol induced promoter, optimized Kozak consensus sequence, and replace native signal sequence against signal peptide of S. cerevisiae alpha factor, avoid restriction sites in synthetic gene which interfere with available vectors and modern standard cloning techniques.

Examples for training (chose one/group): Trigonopsis variabilis D amino acid oxidase:

MAKIVVIGAGVAGLTTALQLLRKGHEVTIVSEFTPGDLSIGYTSPWAGANWLT FYDGGKLADYDAVSYPILRELARSSPEAGIRLINQRSHVLKRDLPKLEGAMS AICQRNPWFKNTVDSFEIIEDRSRIVHDDVAYLVEFASVCIHTGVYLNWLMS QCLSLGATVVKRRVNHIKDANLLHSSGSRPDVIVNCSGLFARFLGGVEDKK MYPIRGQVVLVRNSLPFMASFSSTPEKENEDEALYIMTRFDGTSIIGGCFQP NNWSSEPDPSLTHRILSRALDRFPELTKDGPLDIVRECVGHRPGREGGPRV ELEKIPGVGFVVHNYGAAGAGYQSSYGMADEAVSYVERALTRPNL Human albumine:

MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFA QYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVAT LRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHD NEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVS KLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLL EKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYA RRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNL IKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCK HPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQ LKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL Aspergillus alternative oxidase mitochondrial protein:

MNSLTATAPIRAAIPKSYMHIATRNYSGVIAMSGLRCSGSLVANRHQTAGKR FISTTPKSQIKEFFPPPTAPHVKEVETAWVHPVYTEEQMKQVAIAHRDAKNW ADWVALGTVRMLRWGMDLVTGYRHPPPGREHEARFKMTEQKWLTRFIFLE SVAGVPGMVGGMLRHLRSLRRMKRDNGWIETLLEEAYNERMHLLTFLKLAE PGWFMRLMVLGAQGVFFNGFFLSYLMSPRICHRFVGYLEEEAVITYTRAIKE IEAGSLPAWEKTEAPEIAVQYWKMPEGQRSMKDLLLYVRADEAKHREVNHT LGNLNQAIDPNPYAAKYKDPTKAHPNKGIADLKPTGWEREEVI

 Fungal cellulose:

MIVGILTTLATLATLAASVPLEERQACSSAWGQCGGQNWSGPTCCASGSTC VYSNDYYSQCLPGAASSSSSTRASSTTARASSTTSRSSATPPPGSSTTRVP PVGSGTATYSGNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKV PSFMWLDTFDKTPLMEQTLADIRTANKNGGNYAGQFVVYDLPDRDCAALAS NGEYSIADGGVDKYKNYIDTIRQIVVEYSDIRTLLVIEPDSLANLVTNLGTPKC ANAQSAYLECINYAVTQLNLPNVAMYLDAGHAGWLGWPANQDPAAQLFAN VYKNASSPRALRGLATNVANYNGWNITSPPSYTQGNAVYNEQLYIHAIGPLL ANHGWSNAFFITDQGRSGKQPTGQQQWGDWCNVIGTGFGIRPSANTGDS LLDSFVWIKPGGECDGTSDSSAPRFDSHCALPDALQPAPQAGAWFQAYFV QLLTNANPSFL Prunus dulcis R-HNL5mutant - DNA sequence (advanced training)

synthetic sequence for mature plant protein fused to alpha factor leader sequence of S. cerevisiae: atgagatttccttcaatttttactgctgttttattcgcagcatcctccgcattagctgctccagtcaacactacaac agaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgt tgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgcta aagaagaaggggtatctctcgagaaaagagaggctgaagctcttgccaatacttctgctcatgattttagcta cttgaagtttgtgtacaacgccactgatacaagctcggaaggatcatatgactacattgtaatcggtggagga acatcagggtgtccattggcagcaactttatcagaaaaatacaaggtgcttcttctagaaagaggcactattg ctacagaatacccgaacacgttgactgcagatgggtttgcatataatctgcagcaacaagatgatggaaag acgccagttgaaaggttcgtgtccgaagatggcattgataatgtgcgagccaggatcctcggtggcacgac cataatcaatgcaggcgtctacgccagagctaacatttcattctatagtcaaacaggaattgaatgggacct ggatttggtcaataagacatatgagtgggttgaagacgccattgtggtcaagccaaataatcaatcttggca atctgttataggagagggattcttggaggcgggtattcttccagacaatggatttagtttggatcacgaagcag gaactagactcaccggctcaacttttgacaataatggaacgcgacatgcggctgatgaactgcttaataaa ggagaccctaataacttgctagttgcagttcaggcctcagtagagaagatcctcttctcttccaatacatcaaa tttgtcagctattggagtcatatatacggattctgatggaaactctcatcaggcatttgtacgcggtaacggaga agttattgttagtgcagggacaatcggaacgcctcagcttctactacttagtggcgttggaccagagtcttacc tatcttctctcaacatcacagttgttcagccgaatccttatgttgggcagtttgtgtatgacaatcctcgtaatttcat taatattttgcccccaaatccaattgaagcctctgttgctactgttttaggcattagaagtgattattatcaagtttc tctgtcaagcttgccattttccactccaccctttagtctttttcctacaacatcttaccccctcccaaattcgacttttg

ctcatattgttagccaagttccaggaccattgtctcatggttctgtcacgctaaattcatcatctgacgtgagaat cgctccaaatattaaattcaattactattcaaattccacagaccttgctaattgtgttagcggcatgaagaagct tggtgacttattaaggacaaaggcattagaaccatataaagctcgagatgtgctgggaattgacggtttcaat tatttgggagtacctttgccagagaaccaaacagatgatgcatccttcgaaacattttgtctagataatgtagct tcatactggcattaccacggtggaagccttgttgggaaagtgcttgatgacagtttccgtgttatggggatcaa agcattacgcgttgttgatgcctccactttcccttacgaaccaaacagccatcctcagggcttctatctgatgtta ggaaggtatgtgggccttcaaatcctgcaagaaaggtcaatccggttggaggctattcataatattcaagag tccatgtga

DNA sequence coding for S. cerevisiae alpha factor secretion leader:

atgagatttccttcaatttttactgctgttttattcgcagcatcctccgcattagctgctccagtcaacactacaac agaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgt tgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgcta aagaagaaggggtatctctcgagaaaagagaggctgaagct

Protein sequence of S. cerevisiae alpha factor secretion leader:

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVL PFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA