High-throughput Screening of Soluble Recombinant Proteins Protein Science, 2002, vol 11, 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  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.

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

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

General cloning strategy of HP TargetS-tagHis Protease cleavage site Top10 T-vector pET BL21 Ligation

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

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.

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

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) Eight fusion protein expression vectors

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

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

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

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

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%).

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.

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