Bac-to-Bac expression system

1. Bac-to-Bac expression system Lab of Molecular Genetics 심희연

2. Baculovirus • A family of large rod-shaped viruses • Circular double-stranded genome ranging from 80-180 kbp. • AcMNPV : Autographa califonica multiple nuclear polyhedrosis virus : the best characterized and undergoes a succession of early, late and very late gene expression during its infection cycle. : the strong polyhedrin promoter-> the transcriptional control.

3. Bac-to-Bac Baculovirus Expression System • An efficient site-specific transposition system to generate baculovirus for high-level expression of recombinant proteins • Advantage 1. recombinant virus DNA isolated from selected colonies is not mixed with parental, nonrecombinant virus - Easy colony screening : lacZα gene (Bluo-gal or X-gal) 2. Time-saving expression to identify and purify a recombinant virus

4. site-specific transposition transposed donor vector sequence Bacterial primer sequence

5. Bac-to-Bac Baculovirus Expression System • Used vectors: 1. Donor plasmid vector into which the gene(s) of interest will be cloned 2. Baculovirus shuttle vector (bacmid) : mini-attTn7 target site, lacZ, kanamycin resistance marker 3. Helper plasmid : transposase, tetracycline resistance marker

6. Bacmid -baculovirus shuttle vector AcMNPV bacmid (136kb) • <bacmid> • kanamycin resistance marker • LacZ-mini-attTn7 • ( a short segment containing the attachment site for the bacterial transposon Tn7) • a low-copy-number mini- • Fertility replicon LacZ-mini-attTn7 Kan mini-F • <donor plasmid> • Multiple cloning site • SV40 polyadenylation signal • Tn7R(bacteria transposon) • gusA • Ppolh(promoter) • Gentamicin resistance gene Ppolh Tn7R Tn7R SV40 P(A) gusA Gen Donor plasmid(pFastBac) (6.7kb)

7. Transposon & transpostion • Sequences of DNA that can move around to different positions within the genome of a single cell. • A process called transposition • Transposase: a enzyme that be inserted at another location. • Inverted repeats(IR): a sequence of nucleotides that is the reversed complement of another sequence further downstream.

8. Diagram of the Bac-to-Bac system

9. The END

11. pFastBac1-Transfer vector, donor plasmid • Multiple cloning site • SV40 polyadenylation signal • Tn7L • f1 origin (f1 intergenic region) • Ampicillin resistance gene • pUC origin • Tn7R • Gentamicin resistance gene (complementary strand) • Polyhedrin promoter (PPH)

12. lacZ • The lac gene and its derivatives are amenable to use as a reporter gene in a number of bacterial-based selection techniques such as two hybrid analysis, in which the successful binding of a transcriptional activator to a specific promoter sequence must be determined. • In LB plates containing X-gal, the colour change from white colonies to a shade of blue, corresponds to about 20-100 β-galactosidase units. • Lac operon은 E. coli가 lactose를 galactose와 glucose로 분해하는데 필요한 효소인 beta-galactosidase를 만드는 유전자이다 • IPTG(isopropyl thio-galactoside)를넣어 lac repressor을 유도시킨다.

13. tetracycline

14. <Principle of rapid generation of multigene baculoviruses> • Two transfer vectors, each expressing two genes • recombine with the baculovirus genome maintained in E. coli as a bacmid. • Two genetic loci in the baculovirus genome are used, • recombination is controlled by Tn7 transposition • the bacmid DNA is transfected into insect cells, where it directs expression of all four genes at high levels. • The improved expression characteristics of the eukaryotic expression system promote assembly of correctly processed and folded tetrameric protein complexes.

15. (MNPV) Envelope glycoprotein Envelop protein

16. <Baculovirus life cycle>