engineering magnetosomes to express novel proteins which ones? must be suitable for expressing in...
DESCRIPTION
How to bioengineer a novel protein expression system New host must be able to “read” the sequence Promoters Terminators Capping, splicing and polyA if eukaryote Codon usageTRANSCRIPT
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Engineering magnetosomes to express novel proteinsWhich ones? •Must be suitable for expressing in Magnetospyrillum!•Can’t rely on glycosylation, disulphide bonds, lipidation, selective proteolysis, etc for function!• Best bets are bacterial proteins• Alternatives are eukaryotic proteins that don’t need
any of the above• Short peptides
•Tweaking p18• Linker• Deleting or replacing GFP
•TRZN•Oxalate decarboxylases•Lactate dehydrogenase or other oxalate metab enzyme
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How to bioengineer a novel protein expression system?1.Identify a suitable candidate organism2.DNA must function in this host3.mRNA must function in this host4.Protein must be functional•Must be able to purify large amounts of functional protein
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How to bioengineer a novel protein expression systemNew host must be able to “read” the sequencePromotersTerminatorsCapping, splicing and polyA if eukaryoteCodon usage
![Page 4: Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Cant rely on glycosylation, disulphide](https://reader036.vdocuments.us/reader036/viewer/2022062600/5a4d1bae7f8b9ab0599cbc66/html5/thumbnails/4.jpg)
How to bioengineer a novel protein expression systemNew host must be able to “read” the sequencePromotersTerminatorsCapping, splicing and polyA if eukaryoteCodon usage3. mRNA must function in the new host
![Page 5: Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Cant rely on glycosylation, disulphide](https://reader036.vdocuments.us/reader036/viewer/2022062600/5a4d1bae7f8b9ab0599cbc66/html5/thumbnails/5.jpg)
How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site
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How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site• S-D in prok• Kozak in euk
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How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site• S-D in prok• Kozak in euk• Also needs correct information in 5’ UTR
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How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site• Also needs correct information in 5’ UTR• In eukaryotes also needs 5’ cap & poly-A tail
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How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site• Also needs correct information in 5’ UTR• In eukaryotes also needs 5’ cap & poly-A tail• Must also be properly localized
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How to bioengineer a novel protein expression system?3. mRNA must function in the new host•Must be translated: needs correct ribosome-binding site•Must survive: in euk depends on internal sequences (especially in 3’ UTR) and poly-A tail
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How to bioengineer a novel protein expression system?4. Protein must function in the new host•Must be translated•Must be modified correctly
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How to bioengineer a novel protein expression system?4. Protein must function in the new host•Must be translated•Must be modified correctly• Glycosylation: affects folding, solubility &
localization
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How to bioengineer a novel protein expression system?4. Protein must function in the new host•Must be translated•Must be modified correctly• Glycosylation: affects folding, solubility &
localization• Simple glycosylation can be engineered
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Protein must function in the new hostMust be modified correctlyGlycosylationLipidation•Myristylation (14C) @ N•Palmitoylation (16C)•Isoprenoids
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Protein must function in the new host•Must be modified correctly• Glycosylation• Lipidation• proteolysis
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Protein must function in the new host•Must be modified correctly• Glycosylation• Lipidation• Proteolysis• Disulfide bonds
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Protein must function in the new host•Must be modified correctly• Glycosylation• Lipidation• Proteolysis• Disulfide bonds
Must go to correct location
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Protein must function in the new host•Must be modified correctly• Glycosylation• Lipidation• proteolysis
Must go to correct locationMust survive!
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How to bioengineer a novel protein expression system?3. Protein must function in the new host• Must be modified correctly• Glycosylation• Lipidation• proteolysis
• Must go to correct location• Must survive!
4. Identify suitable gene(s), then obtain in some way, add suitable promoters and terminators, and transform into new host.
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How to bioengineer a novel protein expression system?Hosts?•Escherichia coli
• Pro• Best-understood & fastest growing of all hosts• Most genetic control• Lots of tricks & special-purpose strains
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How to bioengineer a novel protein expression system?Hosts?•Escherichia coli
• Pro• Best-understood & fastest growing of all hosts• Most genetic control• Lots of tricks & special-purpose strains
• Con• Prokaryote: no glycosylation, S-S bonds, etc.
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How to bioengineer a novel protein expression system?Hosts?•Escherichia coli
• Pro• Best-understood & fastest growing of all hosts• Most genetic control• Lots of tricks & special-purpose strains
• Con• Prokaryote: no glycosylation, S-S bonds, etc.
•Saccharomyces cerevisiae (brewer’s yeast)• Pro• Eukaryotic• Fast-growing and well-characterized• Good genetic control• Lots of tricks & special-purpose strains
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Hosts?E. coliS. cerevisiae (brewer’s yeast)• Pro• Eukaryotic• Fast-growing and well-characterized• Good genetic control• Lots of tricks & special-purpose strains
• Con• Does things its own way• Weird glycosylation, etc• Proteins from other eukaryotes often don’t work
when expressed in yeast
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Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris
• Pro• Methylotrophic: grows in [methanol] that kill
most other organisms• Eukaryotic• Fast-growing and well-characterized• Good genetic control with glucose & MeOH• Grows to very high densities
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Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris
• Pro• Methylotrophic: grows in [methanol] that kill
most other organisms• Eukaryotic• Fast-growing and well-characterized• Good genetic control with glucose & MeOH• Grows to very high densities
• Con• Does things its own way• Weird glycosylation, etc• Proteins from other euk often don’t work
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Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells• Pro• Make glycoproteins “faithfully”• Can’t infect plants or animals• Can use promoters of “late genes” to drive
expression of heterologous proteins• Insect cell cultures are easier & more forgiving
than mammalian cell cultures
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Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells• Pro• Make glycoproteins “faithfully”• Can’t infect plants or animals• Can use promoters of “late genes” to drive
expression of heterologous proteins• Insect cell cultures are easier & more forgiving
than mammalian cell cultures• Con• Trickier• Requires cell cultures: expensive!
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Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants
• Pro• Make authentic plant proteins (and sometimes
animal proteins)• Cheap• “Easy” and robust techniques
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Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants
• Pro• Make authentic plant proteins (and sometimes
animal proteins)• Cheap• “Easy” and robust techniques
• Con• Slow• May not process non-plant proteins correctly
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Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants•Animal cell cultures
• Pro• Make authentic animal proteins• “Easy” and robust techniques
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Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants•Animal cell cultures
• Pro• Make authentic animal proteins• “Easy” and robust techniques
• Con• Slow• Expensive!• Purifying proteins can be difficult
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Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants•Animal cell cultures•Transgenic animals
• Pro• Make authentic animal proteins
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Hosts?•E. coli•Saccharomyces cerevisiae (brewer’s yeast)•Pichia pastoris•Baculovirus in cultured insect cells•Transgenic plants•Animal cell cultures•Transgenic animals
• Pro• Make authentic animal proteins
• Con• Slow• Expensive!• Difficult• Purifying proteins can be difficult
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Hosts?Magnetospirillum gryphiswaldense
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Hosts?Magnetospirillum gryphiswaldense•Can propagate plasmids (but pBAM requires pir gene)•Or can insert into chromosome via tnpA (Tn5)-based transposition: no variation in expression due to copy number or growth stage
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Hosts?Magnetospirillum gryphiswaldense•Borg optimised rbs, promoter & codon usage
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Hosts?Magnetospirillum gryphiswaldense•Borg optimised rbs, promoter & codon usage•Developed inducible system based on tetracycline
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Hosts?Magnetospirillum gryphiswaldense•Borg optimised rbs, promoter & codon usage•Developed inducible system based on tetracycline•Fuse protein to C-terminus of mamC
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Hosts?Magnetospirillum gryphiswaldense•Borg optimised rbs, promoter & codon usage•Developed inducible system based on tetracycline•Fuse protein to mamC C-terminus: exposed at surface
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Hosts?Magnetospirillum gryphiswaldense•Borg optimised rbs, promoter & codon usage•Developed inducible system based on tetracycline•Fuse protein to mamC C-terminus: exposed at surface•Purify with magnets
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Assignment•Design a mamC C-terminal protein fusion
• Design DNA sequence encoding a useful protein
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Assignment•Design a mamC C-terminal protein fusion•Design DNA sequence encoding a useful protein•Replace eGFP of pJH3 with your protein
• Best to use MluI and NheI sites
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AssignmentBest to use MluI and NheI sitesDesign oligos that add MluI in frame at 5” end and NheI at 3’end
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AssignmentBest to use MluI and NheI sitesDesign oligos that add MluI in frame at 5’ and NheI at 3’endDigest vector & clone with MluI and NheI then ligate
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AssignmentBest to use MluI and NheI sitesDesign oligos that add MluI in frame at 5’ and NheI at 3’endDigest vector & clone with MluI and NheI then ligateFind & analyze clones
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Transcription
Prokaryotes have one RNA polymerase
makes all RNA
core polymerase = complex of 5 subunits (’)
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Transcription
Prokaryotes have one RNA polymerase
makes all RNA
core polymerase = complex of 5 subunits (’)
not absolutely needed, but cells lacking are very sick
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Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous
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Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity
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Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters
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Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters• Different sigmas bind different promoters
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Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters3) Once bound, RNA polymerase “melts” the DNA
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Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template
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Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template5) RNA polymerase catalyzes phosphodiester
bonds, melts and unwinds template
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Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template5) RNA polymerase catalyzes phosphodiester
bonds, melts and unwinds template6) sigma falls off after ~10 bases are added
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Structure of Prokaryotic promotersThree DNA sequences (core regions)
1) Pribnow box at -10 (10 bp 5’ to transcription start)5’-TATAAT-3’ determines exact start site: bound by factor
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Structure of Prokaryotic promotersThree DNA sequences (core regions)
1) Pribnow box at -10 (10 bp 5’ to transcription start)5’-TATAAT-3’ determines exact start site: bound by factor
2)” -35 region” : 5’-TTGACA-3’ : bound by factor
![Page 58: Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Cant rely on glycosylation, disulphide](https://reader036.vdocuments.us/reader036/viewer/2022062600/5a4d1bae7f8b9ab0599cbc66/html5/thumbnails/58.jpg)
Structure of Prokaryotic promotersThree DNA sequences (core regions)
1) Pribnow box at -10 (10 bp 5’ to transcription start)5’-TATAAT-3’ determines exact start site: bound by factor
2)” -35 region” : 5’-TTGACA-3’ : bound by factor3) UP element : -57: bound by factor
![Page 59: Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Cant rely on glycosylation, disulphide](https://reader036.vdocuments.us/reader036/viewer/2022062600/5a4d1bae7f8b9ab0599cbc66/html5/thumbnails/59.jpg)
Structure of Prokaryotic promotersThree DNA sequences (core regions)
1) Pribnow box at -10 (10 bp 5’ to transcription start)5’-TATAAT-3’ determines exact start site: bound by factor
2)” -35 region” : 5’-TTGACA-3’ : bound by factor3) UP element : -57: bound by factor
![Page 60: Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Cant rely on glycosylation, disulphide](https://reader036.vdocuments.us/reader036/viewer/2022062600/5a4d1bae7f8b9ab0599cbc66/html5/thumbnails/60.jpg)
Structure of Prokaryotic promotersThree DNA sequences (core regions)
1) Pribnow box at -10 (10 bp 5’ to transcription start)5’-TATAAT-3’ determines exact start site: bound by factor
2)” -35 region” : 5’-TTGACA-3’ : bound by factor3) UP element : -57: bound by factorOther sequences also often influence transcription! Eg CAP site in lac promoter
![Page 61: Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Cant rely on glycosylation, disulphide](https://reader036.vdocuments.us/reader036/viewer/2022062600/5a4d1bae7f8b9ab0599cbc66/html5/thumbnails/61.jpg)
Termination of transcription in prokaryotes
1) Sometimes go until ribosomes fall too far behind
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Termination of transcription in prokaryotes
1) Sometimes go until ribosomes fall too far behind
2) ~50% of E.coli genes require a termination factor called “rho”
![Page 63: Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Cant rely on glycosylation, disulphide](https://reader036.vdocuments.us/reader036/viewer/2022062600/5a4d1bae7f8b9ab0599cbc66/html5/thumbnails/63.jpg)
Termination of transcription in prokaryotes
1) Sometimes go until ribosomes fall too far behind
2) ~50% of E.coli genes require a termination factor called “rho”
3) Our terminator (rrnB) first forms an RNA hairpin, followed by an 8 base sequence TATCTGTT that halts transcription