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
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.