In vitro expression of In vitro expression of BVDV capsid proteinBVDV capsid protein

Corpus Christi College, University of OxfordCorpus Christi College, University of OxfordGlycobiology Institute, Department of BiochemistryGlycobiology Institute, Department of Biochemistry

KOR SHU CHANKOR SHU CHAN

ObjectivesObjectives

• Construct an expression vector for the part of the capsid region of the BVDV genome encoding the first 84 amino acids of the capsid protein in pIVEX2.4d and pIVEX2.3d

• Express it in vitro in bacterial lysate (RTS, Roche) to identify suitable conditions for expression

Bovine viral diarrhoea virus Bovine viral diarrhoea virus (BVDV)(BVDV)

• Pestivirus• May cause diarrhoea, mucosal

disease and severe haemorrhagic conditions in calves

• May lead to calf pneumonia, infertility and various birth abnormalities

• Used as a surrogate model for HCV

BVDV capsid proteinBVDV capsid protein

… tcc gac aca aaa gat gaa ggg gtg gtg agg aag aag caa caa aag cca gat agg ttg gaa aag ggg aga atg aag ata aca cct aag gag tca gag aaa gac agt aag acc aag ccg cca gat gct acg ata gtg gta gat gga gtc aag tat cag gta aag aaa aaa gga aaa gtc aag agc aag aac acc cag gac ggc tta tac cac aac aaa aat aaa cct caa gag tcg cgc aag aaa cta gag aaa gcc cta ttg gcc …

BV

DV

capsid

regio

n

sequ

ence

5’ 3’structural Non-structural

BVDV capsid proteinBVDV capsid protein

… tcc gac aca aaa gat gaa ggg gtg gtg agg aag aag caa caa aag cca gat agg ttg gaa aag ggg aga atg aag ata aca cct aag gag tca gag aaa gac agt aag acc aag ccg cca gat gct acg ata gtg gta gat gga gtc aag tat cag gta aag aaa aaa gga aaa gtc aag agc aag aac acc cag gac ggc tta tac cac aac aaa aat aaa cct caa gag tcg cgc aag aaa cta gag aaa gcc cta ttg gcc …

• 100 amino acids• Last 16 are

transmembrane• Attempt to

express the first 84 amino acids, the part of the protein thought to be water-soluble

pIVEX2.4d vs. pIVEX2.3dpIVEX2.4d vs. pIVEX2.3d

Rapid Translation System Rapid Translation System (RTS)(RTS)

• Bacterial lysate as expression medium

• Easy to use: no host-cell transformation, culturing or lysis necessary

• Fast: each expression takes 4-6 hours• High efficiency: up to 20 µg protein

per 50 µl in four hours

ResultsResults

pIVEX2.4dpIVEX2.4d

4k3k2k

Digested and CIP-treated pIVEX2.4d

PCR fragments generated from cp7 template

400200

undigested

200

400

digested

pIVEX2.4dpIVEX2.4dLigation-transformation

pIVEX2.4dpIVEX2.4d

4k3k2.5k2k1.5k1k800

Ph

osp

ho

rylated

pIV

EX

2.4dD

eph

osp

ho

rylated

pIV

EX

2.4dHindIII digestion of Maxiprep products

8001k1.5k2.5k3k

400200

400200

PCR screening

400200

400200

pIVEX2.4dpIVEX2.4d

1 2 3 4 GFP (control)

SAMPLES

pIVEX2.3dpIVEX2.3d

200

400

200

400

w/o template w/o forward primer w/o reverse primer

ConclusionsConclusions• Gene desired amplified by PCR using primers

designed for pIVEX2.4d but not those designed for pIVEX2.3d

• Ligation-transformation successful using pIVEX2.4d

• Expression attempted

• Anti-penta-His-tag (with HRP moiety) can be used to detect the presence of the desired protein

• Revise PCR protocol for pIVEX2.3d (e.g. redesign primers, change reaction conditions)

AcknowledgementsAcknowledgements• Dr. Devanagoud Patil• Dr. Narayan Ramamurthy• Dr. Steve Woodhouse• Dr. Nicole Zitzmann• All other people in the Virus lab and

Proteomics lab• With thanks to Prof. Raymond Dwek