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High-throughput Screening of Soluble Recombinant Proteins

High-throughput Screening of Soluble Recombinant Proteins Protein Science, 2002 , vol 11 , 1714-1719 YAN-PING SHIH,1 WEN-MEI KUNG,1 JUI-CHUAN CHEN, CHIA-HUI YEH,ANDREW H.-J. WANG Speaker: Chung-Sheng Liu 2002/10/29. Introduction.

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High-throughput Screening of Soluble Recombinant Proteins

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  1. High-throughput Screening of Soluble Recombinant Proteins Protein Science, 2002 , vol 11 , 1714-1719 YAN-PING SHIH,1 WEN-MEI KUNG,1 JUI-CHUAN CHEN, CHIA-HUI YEH,ANDREW H.-J. WANG Speaker: Chung-Sheng Liu 2002/10/29

  2. Introduction • The function of a gene is manifested by the protein it encodes. • Genome sequencing of many organisms has led to the concept of analyzing protein function on a genome-wide scale. • Structural genomics and proteomics, therefore, have become major research foci.

  3. Cloning and expression in Escherichia coli • Advantage： -has relatively simple genetics, is well characterized - has a relatively rapid growth rate - has few post-translational protein modifications • Disadvantage： - expressing heterologous proteins in E.coli are frequently expressed as insoluble aggregated folding intermediates, known as inclusion bodies

  4. Blunt-EndPCR PCR product 5’AATTC CTCGA3’ 3’TTAAG GAGCT5’ Enzyme digestion 5’AATTC C3’ 3’G GAGCT5’ Subcloning

  5. His S-tag Target Protease cleavage site pET Top10 T-vector Ligation BL21 General cloning strategy of HP

  6. Sticky-End PCR: New Method for Subcloning Ligation with vectors which had been double digested and were dephosphorylated by calf intestine alkaline phosphatase T4 polynucleotide kinase + ATP ~25% final product carries two cohesive ends

  7. Sticky-end PCR and directional cloning methods’ advantages • Simpler: It allows direct cloning of PCR products into multiple expression vectors. • It is more accurate in theory and also in practice.

  8. Eight different fusion protein expression vectors and Three type host strains JM109(DE3): both plasmid preparation and protein expression BL21-Gold(DE3) or -CondonPlus(DE3): alleviate codon bias or toxicity

  9. Eight fusion protein expression vectors • His (histidine) : pET-28a (Novagen) • Trx (thioredoxin) : pET-32a (Novagen) • NusA (NusA protein) : pET-43.1a (Novagen) • CAP (cellulose-associated protein) : pET-35b2 (Novagen) • CBP (calmodulin binding protein) : pET-22b+ (Novagen) • Intein (chitin binding tag) : pTYB11(NEB) • MBP (maltose-binding protein) : pMAL-C2XC (NEB) • GST (glutathione S-transferase) : pGEX-4T (Pharmacia)

  10. ~40 genes into eight expression vectors ( >300 cloning reactions) >95% success cloning rate >80% highly express and soluble these target protein: 9-100kD

  11. Fusion Proteins Solubility Test Lane 1: whole cell lysates of induced cells Lane 2: whole cell lysates of uninduced cells Lane 3: soluble proteins with induction

  12. Statistical Analysis of Soluble Protein Ratio NusA(54kD): 60% MBP (42kD): 60% GST (24kD): 38% #90,000g ultracentrifugal force: eliminate partially folded protein aggregates

  13. N terminal- -C terminal Tag Target His Protease cleavage site Two steps of affinity purification fusion protein: 5~20mg/l LB >90% purity

  14. Summary • No restriction digestion of the PCR products. • All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%).

  15. Summary • 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. • High-speed centrifugation in a 96-tube format is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis.

  16. Thanks for your attention

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