BCMB20005 Lecture Notes - Lecture 10: Elution, Galactose, Escherichia Coli
2 Aug 2018
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Lecture 10
Protein expression systems PART I
Recall from lecture 9: Key steps in producing recombinant proteins
Bacterial expression systems RECALL
Requirements of a bacterial expression system:
1. Expression vector (plasmid)
Sequences for expression and regulation of expression
2. Host bacterial cells (often modified)
Important properties:
i. Regulate expression
ii. Provide suitable environment for expression and correct folding
Requirements for high level expression
● A strong regulated promoter:
○ able to make large numbers of mRNA transcripts - vector
○ able to produce mRNA transcripts when required - vector
● Promoter must suit to host cell, ribosome binding site on the expression vector just
before the coding region for transcription and translation
● Effective translation of proteins from mRNA transcript s - host cells
● Correct processing and folding of the translated proteins - host cells
Regulating gene expression: the lac
operon
Lac
operon: collection of three genes required for lactose transport and metabolism in E.coli
● Adjacent structural genes: lacZ
(β-galactosidase), lacY
(lactose permease) and lacA
(thiogalactosidase transacetylase)
● Allows for the effective digestion of lactose in vivo
●LacY
- Lactose permease (cytoplasmic membrane) transports lactose into cell
●LacZ
-
β - galactosidase (cytoplasm) cleaves lactose into glucose and galactose
● LacI gene upstream of the promoter region that encodes the lac repressor protein
● If no lactose is present, lac
genes are turned off, no enzyme production, therefore
energy is saved
○ lacI gene is always expressed, always producing repressor protein that binds
to lacO, RNA polymerase cannot interact at the promoter site
● Genetic switch turns genes on and off = REGULATION
Control mechanism:
● Lac repressor (regulatory protein) encoded by lacI gene
● LacI gene is constitutively expressed
● In absence of lactose, Lac repressor binds tightly to operator sequence
○ LacI is a tetramer/ a dimer of dimer
● It binds to a specific sequence of 24 nucleotides of that lac operator sequence
● Lac repressor interferes with binding of RNAP to promoter
↳Prevents gene transcription by RNA polymerase
● In presence of lactose (or allolactose):
○ Lac repressor cannot bind operator sequence due to structural/conformational
changes caused by lactose binding to the repressor protein
● Consequence:
○ Lac repressor no longer interferes with binding of RNAP to promoter
↳LacZ, Y, A
genes are transcribed
Lactose and allolactose are inducers when it act and Interact with lac repressor protein,
therefore promotes transcription of gene that is controlled by repressor protein.
Using the lac
repressor in vitro
● Can exploit this system for controlled regulation of protein expression in vitro
● Need to provide:
○ Lacl gene encodes lac repressor protein
○ LacI gene plus promoter and operator sequence RNA - expression vector
○ Plus an inducer molecule: IPTG (analogue of lactose)
■ When lac repressor is bind to the operator sequence, inducer binds to
lac repressor and release from operator
■ Not a substrate of beta galactosidase
■ Use bacterial expression vector (e.coli), already have lac operon, add
IPTG, it is already of lactose, an analogue when if it can be digested
by Beta galactosidase, other genes from lac operons produced, beta
thioglucosidase break down that molecules
■ That’s why we don't use lactose, beta galactosidase break down the
lactose by production of other genes from E.coli
■ IPTG not a substrate of beta , maintain in cell for constant level. Can
turn this on or off.
Inducing protein expression in vitro
● LacI gene expressing, lac tetramer always freely diffusing
● Tetramer binds to the 24 nucleotides in operator, preventing RNA Polymerase from
interacting with the promoter
● Genes downstream will not be transcribed
Document Summary
Recall from lecture 9: key steps in producing recombinant proteins. Requirements of a bacterial expression system: expression vector (plasmid, host bacterial cells (often modified) Provide suitable environment for expression and correct folding i. ii. Able to make large numbers of mrna transcripts - vector. Able to produce mrna transcripts when required - vector. Promoter must suit to host cell, ribosome binding site on the expression vector just before the coding region for transcription and translation. Effective translation of proteins from mrna transcript s - host cells. Correct processing and folding of the translated proteins - host cells. Regulating gene expression: the lac operon (thiogalactosidase transacetylase) Lac operon: collection of three genes required for lactose transport and metabolism in e. coli. Adjacent structural genes: lacz ( -galactosidase), lacy (lactose permease) and laca. Allows for the effective digestion of lactose in vivo. Lacy - lactose permease (cytoplasmic membrane) transports lactose into cell. Lacz - - galactosidase (cytoplasm) cleaves lactose into glucose and galactose.
