In vitro expression of BVDV capsid protein Corpus Christi College, University of Oxford Glycobiology Institute, Department of Biochemistry KOR SHU CHAN

Objectives 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 (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 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 … BVDV capsid regionsequence 5’3’ structuralNon-structural

BVDV 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. pIVEX2.3d

Rapid Translation System (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 µ g protein per 50 µ l in four hours

Results

pIVEX2.4d 4k 3k 2k Digested and CIP- treated pIVEX2.4d PCR fragments generated from cp7 template undigested digested

pIVEX2.4d Ligation-transformation

pIVEX2.4d 4k 3k 2.5k 2k 1.5k 1k 800 Phosphorylated pIVEX2.4d Dephosphorylated pIVEX2.4d HindIII digestion of Maxiprep products 800 1k 1.5k 2.5k 3k PCR screening

pIVEX2.4d GFP (control) SAMPLES

pIVEX2.3d w/o templatew/o forward primerw/o reverse primer

Conclusions 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)

Acknowledgements 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