Recombinant Protein Expression Retroviral system

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Octavia Goodwin
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1 Recombinant Protein Expression Retroviral system

2 Viruses Contains genome DNA or RNA Genome encased in a protein coat or capsid. Some viruses have membrane covering protein coat enveloped virus Ø Essential Cell Biology: pages

3 How does a virus work? General life cycle Proteins on surface determine host specificity Virus uses host resources Some viruses carry RNA, not DNA. Some viruses insert DNA into host chromosome.

4 Types of viruses

5 Viral genome Can be DNA (can be circular) or RNA protein capsid protease integrase polymerase Examples of genes/proteins

6 Retrovirus (RNA) RNA is converted to DNA; then, DNA is transcribed à translated

7 Integration of Retroviral DNA into host RNA à DNA à Host DNA Viral DNA in host cell

8 Insertion of Viral DNA into host Chromosome DNA virus Host DNA integrated viral DNA Host DNA Host Viral chromosome DNA in host cell

9 Diagram of retrovirus determine species infected

10 Retrovirus genome LTR gag-pol env LTR RNA packaging signal

11 Retroviral expression system - MLV System from murine leukemia virus (MLV) Recombinant cdna is introduced into cell by infection much more efficient than transfection. Cells have to be dividing for infection Recombinant cdna can be integrated into host genome stable. Create modified genome in host

12 Retroviral expression system - MLV Two part system 1. Cell line with part of viral genome integrated into chromosome. Integrated retroviral DNA is missing signal for packaging RNA into virus. Cannot form new virus particle. 2. Plasmid vector with: cdna to be expressed. RNA packaging signal

13 Normal viral DNA in host LTR LTR RNA packaging signal LTR = long terminal repeat part of integration mechanism contains transcriptional enhancer

14 Packaging cell line Replication incompetent viral DNA in packaging cell line No RNA packaging signal Provides all the proteins to make viral particle RNA cannot be packaged in virus Ecotropic packaging virus will not infect host

15 Retroviral expression vector LTR RNA packaging signal cdna to express LTR The expression vector does not have any viral genes for replication, coat formation cannot produce virus.

16 Packaging cell viral proteins RNA without packaging signal X no virus

17 Virus production retroviral vector packaging cell transfect integration transcription Virus RNA w/packaging signal contains vector sequence

18 Retroviral expression system Transected packaging cell line produces virus containing the RNA transcribed from the vector DNA. The recombinant virus can transduce other cells. The transduced vector will not replicate no new virus

19 Retroviral expression system

20 Transduction host What type of cell will a virus infect tranduce In retroviral expression: Host determined by envelope protein in packaging cell Amphotropic will transduce most cell types Ecotropic infectivity restricted to a few species envelope protein

21 Advantages of viral expression systems More efficient than transfection - ~100% Stable integration of infected cdna into host.

22 Lentivirus A type of retrovirus HIV Can infect dividing and non-dividing cells Integrates at random position in genome, may cause mutation

23 Immunoprecipitation

24 Immunoprecipitation original version Add polyethylene glycol (PEG)

25 Immunoprecipitation - modern version

26 Step 1- bind Ab to Protein A-Sepharose Protein A found in the cell wall of Staphylococcus aureus Binds to immunoglubulins disrupts immune response in host. Used in vitro to bind IgG purification of Ab, IP Protein G similar IgG binding protein from Streptococcal bacteria

27 Step 1- bind Ab to Protein A-Sepharose Protein A binds to Fc region of IgG Why is this important in IP? Protein A-IgG complex

28 Step 1- bind Ab to Protein A-Sepharose bead + Ab() bead Protein A covalently bound to Sepharose centrifuge supernatant pellet bead

29 Step 2 incubate w/sample + bead bead Protein sample Protein A-sepharose/Ab centrifuge Pellet beads with Ab/Ag, some sup remains Supernatant unbound protein bead

30 Step 3 wash beads bead Pellet beads with Ab/Ag, no sample remains bead 1. suspend in wash buffer 2. centrifuge 3. repeat 2X Supernatant unbound protein

31 Immunopreciptation - assay Elute and load onto gel - Coomassie or silver stain - Western blot for antigen - Western blot for associated protein Assay activity - enzyme

32 IP à Western blot IP Ab can show up on Western blot Why? Heavy chain = kdalton Light Chain = 25 kdalton

33 IP à Western blot Rabbit IP Ab Mouse IP Ab Blot with: - Mouse 1 o Ab - a-mouse 2 o Ab 50 kda 40 kda 30 kda 20 kda

34 IP à Western blot GFP Rabbit IP Ab Mouse IP Ab Blot with: - Mouse 1 o Ab - a-mouse 2 o Ab 50 kda 40 kda 30 kda 20 kda

35 Is binding specific? Include a negative control: protein sample + non-specific Ab (same species and type as specific Ab) Reduce non-specific Use good 1 o Ab Wash beads well Use salt in buffer mm NaCl

36 Negative control - clean IP Rabbit a-gfp IP Rabbit nonspecific Blot with: - Mouse 1 o Ab - a-mouse 2 o Ab 50 kda 40 kda 30 kda 20 kda

37 Negative control some background IP Rabbit a-gfp IP Rabbit nonspecific Blot with: - Mouse 1 o Ab - a-mouse 2 o Ab 50 kda 40 kda 30 kda 20 kda

38 Cdk s associate with Cyclins cdc25 One phosphorylation site activates; one inhibits 38

39 Co-IP à Western blot IP a-rabbit Cyclin IP nonspec Ab IP a-rabbit Cyclin IP nonspec Ab Blot w/a- Cyclin Ab Blot w/a- Cdk Ab 50 kda 40 kda 50 kda 40 kda 30 kda 20 kda 30 kda 20 kda

40 Identify TF binding site in genome: ChIP - Seq

41 Chromatin Immunoprecipitation (ChIP)

42 ChIP

VIROLOGY. Engineering Viral Genomes: Retrovirus Vectors

VIROLOGY. Engineering Viral Genomes: Retrovirus Vectors VIROLOGY Engineering Viral Genomes: Retrovirus Vectors Viral vectors Retrovirus replicative cycle Most mammalian retroviruses use trna PRO, trna Lys3, trna Lys1,2 The partially unfolded trna is annealed