High-throughput Screening of SolubleRecombinant Proteins

Protein Science, 2002 , vol 11 , 1714-1719YAN-PING SHIH,1 WEN-MEI KUNG,1 JUI-CHUAN CHEN,

CHIA-HUI YEH,ANDREW H.-J. WANG

Speaker: Chung-Sheng Liu2002/10/29

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-tagHisProtease cleavage site

Top10T-vector

pET

BL21Ligation

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: 9-100kD

Lane 1: whole cell lysates of induced cellsLane 2: whole cell lysates of uninduced cellsLane 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 purificationTag Target His

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.

Thanks for your attention