Cloning and Expression of Recombinant Proteins

Transcription

1 Cloning and Expression of Recombinant Proteins Dr. Günther Woehlke Dept. Physics E22 (Biophysics) Technical University Munich James-Franck-Str. D Garching Germany 1 Created May 31, 2013

2 Bacterial plasmids 2 May 29, 2013, the question how DNA can be shuffled between organisms for biotech purposes caused a lot of speechlessness... I concluded that it is helpful to review the process systematically.

3 ! 90,000 bp 99 coding sequences 3 Natural plasmid are huge and contain a multitude of genes. E.g., the plasmid associated with the entero-hemolytic E. coli strain O157 is more than 90 kb (kilo-basepairs) long and contains genes for its on replication, transduction into an acceptor strain, pili, pathogeneity factors, and other.

4 2686 bp < 5 coding sequences natural plasmid stripped to minimal elements 4 Natural plasmid are huge and contain a multitude of genes. For biotechnological purposes, these plasmids have been stripped down to their essentials.

5 2686 bp < 5 coding sequences origin of replication 5 Plasmids use in biotech have been stripped down to their essentials. The minimal requirement comprises: the origin of replication (in most cases ColEI, which works by priming of DNA synthesis through RNA II and its counter-acting anti-sense RNA I, regulated by the 4-helix protein rop), 1. Eguchi Y, Itoh T, Tomizawa J (1991) Antisense RNA. Annu Rev Biochem 60: doi: /annurev.bi

6 2686 bp < 5 coding sequences origin of replication 6 Plasmids use in biotech have been stripped down to their essentials. The minimal requirement comprises: the origin of replication (in most cases ColEI, which works by priming of DNA synthesis through RNA II and its counter-acting anti-sense RNA I, regulated by the 4-helix protein rop), 1. Eguchi Y, Itoh T, Tomizawa J (1991) Antisense RNA. Annu Rev Biochem 60: doi: /annurev.bi

7 β-lactamase (confering ampr) 2686 bp < 5 coding sequences 7 Plasmids use in biotech have been stripped down to their essentials. The minimal requirement comprises: the origin of replication, an antibiotics resistance gene (here: the ampicillin resistance, based on a protein called beta-lactamase),

8 multiple Bacterial plasmids β-lactamase (confering ampr) 2686 bp < 5 coding sequences origin of replication cloning site 8 Plasmids use in biotech have been stripped down to their essentials. The minimal requirement comprises: the origin of replication, an antibiotics resistance gene (here: the ampicillin resistance, based on a protein called beta-lactamase), a multiple cloning (sometimes also called polycloning ) site,

9 β-lactamase (confering ampr) 2686 bp < 5 coding sequences marker (optional) origin of replication 9 Plasmids use in biotech have been stripped down to their essentials. The minimal requirement comprises: the origin of replication, an antibiotics resistance gene (here: the ampicillin resistance, based on a protein called beta-lactamase), a multiple cloning (sometimes also called polycloning ) site, and (optional) selection markers that help identifying correct clones.

10 marker (optional) multiple Bacterial plasmids β-lactamase (confering ampr) 2686 bp < 5 coding sequences origin of replication cloning site 10 Plasmids use in biotech have been stripped down to their essentials. The minimal requirement comprises: the origin of replication, an antibiotics resistance gene (here: the ampicillin resistance, based on a protein called beta-lactamase), a multiple cloning (sometimes also called polycloning ) site, and (optional) selection markers that help identifying correct clones.

11 Plasmids (symbolic, see right) bp size multiple, 1-200/cell) Chromosome ~ bp size (symbolic;1/cell) 11

12 Plasmids (symbolic, see right) bp size multiple, 1-200/cell) Chromosome ~ bp size (symbolic;1/cell) Membrane Lipids Proteins Carbohydrates 12

13 Plasmids (symbolic, see right) bp size multiple, 1-200/cell) Membrane Lipids SDS sodium docecyl sulfate Chromosome ~ bp size (symbolic;1/cell) Proteins SDS NaOH, ph!10 Carbohydrates 12

14 Plasmids Bacterial plasmids SD S NaOH, ph!10 plasmid isolation or preparation Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7: insoluble aggregate

15 Plasmids Bacterial plasmids transformation Ca 2+, Mn 2+,... heat shock 42 C,electroporation Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:

16 SacI BamHI Bacterial plasmids My Coding Sequence of Interest GAGCTC GGATCC GGATCC GAGCTC polycistronic RNA 15 Natural plasmid are huge and contain a multitude of genes. For biotechnological purposes, these plasmids have been stripped down to their essentials.

17 SacI BamHI Bacterial plasmids My Coding Sequence of Interest C G GATCC GAGCT polycistronic RNA 16 Natural plasmid are huge and contain a multitude of genes. For biotechnological purposes, these plasmids have been stripped down to their essentials.

18 SacI BamHI Bacterial plasmids My Coding Sequence of Interest C G GATCC GAGCT polycistronic RNA Phosphate Base Ribose G AGCT C G AGCT C 17 Natural plasmid are huge and contain a multitude of genes. For biotechnological purposes, these plasmids have been stripped down to their essentials.

19 SacI BamHI Bacterial plasmids My Coding Sequence of Interest C G GATCC GAGCT polycistronic RNA Base Ribose G AGCT C G AGCT C 18 Natural plasmid are huge and contain a multitude of genes. For biotechnological purposes, these plasmids have been stripped down to their essentials.

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21 NaOH, ph 10 Plasmids (symbolic, see right) bp size multiple, 1-200/cell) Chromosome ~ bp size (symbolic;1/cell) 21

22 NaOH, ph 10 Plasmids (symbolic, see right) bp size multiple, 1-200/cell) Chromosome ~ bp size (symbolic;1/cell) 21