tecnologie ricombinanti avanzate a.a. 2016/2017 · cellule di insetto (baculovirus) cellule di...

TECNOLOGIE RICOMBINANTI AVANZATE

A.A. 2016/2017

→  production of proteins from cloned genes

→  protein & metabolic engineering

Elisabetta BERGANTINO [email protected]

32 hours, from Monday 27 february to Thursday 27 april

→  design and production of antibodies

Marina DE BERNARD [email protected]

16 hours, from Thursday 4 may

•  MOLECULAR BIOTECHNOLOGY Glick, Pasternak, Patten – 4th edition American Society for Microbiology Press •  appunti di lezione e articoli forniti

•  esame mediante accertamento scritto 6 domande aperte (4 Bergantino + 2 De Bernard)

Password: Mullis85

IL DNA RICOMBINANTE NON SERVE SOLO PER STUDIARE I GENI

PRODUZIONE DI PROTEINE PER MEZZO DELL’ INGEGNERIA GENETICA

-Proteine d’interesse scientifico: es., CRISTALLIZZAZIONE. -PROTEINE DI INTERESSE TERAPEUTICO. -PROTEINE DI INTERESSE COMMERCIALE (ENZIMI). -PROTEINE DA UTILIZZARE COME ANTIGENI PER LA PRODUZIONE DI ANTICORPI POLICLONALI E MONOCLONALI. -REAGENTI PER LA RICERCA DI BASE E APPLICATA.

A cosa possono servire le proteine ricombinanti?

Sistemi di espressione

Procariotici (E.Coli)

Eucariotici:

Saccharomyces Cerevisiae Pichia Pastoris

Cellule di insetto (Baculovirus)

Cellule di mammifero in coltura (CHO etc.)

Animali transgenici

Piante transgeniche

Sintesi di proteine cell-free Ingegneria proteica

Expression of recombinant proteins in E.coli

Genes in PROKARYOTES may have - constititive expression - regulated expression (ex. Lac operon) When heterologous proteins are produced in bacteria Strong and regulated promoters are ususlly used Continuous production causes: - inhibition of cellular functions - energy loss - plasmid loss (IF plasmids are used !!)

Factors affecting expression of cloned

Expression of eukaryotic gene in bacteria

Basic structure of an expression plasmid

- replication origin;

-  selection marker;

-  strong and inducible promoter;

-  ribosome binding site;

-  (ORF coding for a tag or a fusion partner, w/wo proteolytic cleavage site);

-  unique restriction sites;

-  transcription terminator.

The aim is to maximize the yield of the expressed heterologous protein The sole cloning of the gene of interest (GoI) does not garantee a good level of expression of the encoded protein;

It could be necessary to modify the characteristics of (i) the expression vector chosen for cloning, (ii ) the cloned gene and/or (iii ) the protein under study.

(i ) Characteristics of the expression vector

•  Number of copies and stability

•  15-60 copies / cell (plasmidd derived from pMB1/ColE1) •  100-200 copies / cell (pUC serie and derivedplasmids) •  10-12 copies / cell (p15A replicon) → co-expression of proteins

•  Need for selective pressure (antibiotic) •  Vectors containing genes or operators essential for the cell (complementation) •  Clonage into the bacterial chromosome

•  Promoters •  lac regulated by the repressor lacI and induced by IPTG. A good

concentration of lacI is necessary (allels lacIq or lacIq1 present in the vector, up mutations, 10X affinity). lacUV5 (2 mutations in the -10 region, 1 at -66, CAP), frequently used since its regulation is independent from cAMP.

•  trp regulated by the repressor trpR. Vectors based on it can be used in all strains; easy induction by sustraction of triptophane from the medium (not adequate for proteins rich in Trp).

•  tac and trc hybrid promoters (1 nucl. ≠, regions –10 of lac and –35 of trp, DCAP). Induced byIPTG (generally from 0.5 to 4 mM), stronger than Plac and Ptrp.

•  araBAD (Invitrogen) induced by L-arabinose, repressed by glucose; economic system, expression level controlled by sugar concentration; however, in non-saturating concentrations, heterogeneity in the cell population is observed.


(when arabinose is present)

(no arabinose)

•  Promoters •  lac regulated by the repressor lacI and induced by IPTG. A good

concentration of lacI is necessary (allels lacIq or lacIq1 present in the vector, up mutations, 10X affinity). lacUV5 (2 mutations in the -10 region, 1 at -66, CAP), frequently used since its regulation is independent from cAMP.

•  trp regulated by the repressor trpR. Vectors based on it can be used in all strains; easy induction by sustraction of triptophane from the medium (not adequate for proteins rich in Trp).

•  tac and trc hybrid promoters (1 nucl. ≠, regions –10 of lac and –35 of trp, DCAP). Induced byIPTG (generally from 0.5 to 4 mM), stronger than Plac and Ptrp.

•  araBAD (Invitrogen) induced by L-arabinose, repressed by glucose; economic system, expression level controlled by sugar concentration; however, in non-saturating concentrations, heterogeneity in the cell population is observed.

•  λ pL regulated by the repressor of λ cI; the conditional mutant cI857 is used, temperature- sensible (wt at 28-32°C, inactive at 42C).

•  cspA cold-shock protein of E.coli (repressed at 37°C, active at 10°C).

(i ) Characteristics of the expression vector “leaky” promoters !!

•  Promoter of gene 10 of phage T7

•  All genes of phage T7 are recognized and trnscribed by the T7 RNA polymerase (the product of gene 10 -major capsid protein- is the most rapidly sinthezized during infection);

•  T7 RNA polymerase (gene 1 of T7) has a stringent specificity for its own promoters and very high processivity (5÷10 times higher than RNA Pol of E.coli);

•  The gene coding T7 RNA has been introduced into the chromosome of E.coli (into the pro-phage λDE3), under the control of the promoter lacUV5;

•  The gene of interest is cloned into the plasmid, under the control of the promoter of gene 10 of T7.


lac pro T7 RNA Pol

g10 pro Gene of interest

desired protein

Inducer (IPTG)

T7 RNA polymerase

Vectors of pET serie (Rosenberg et al, 1987, Gene) - Invitrogen


•  Promoter of gene 10 of phage T7 • 

•  Promoter of gene 10 of phageT7

•  High levels of transcription and expression (up to 40-50% of total proteins). Not without disavantages: o  excessive levels of mRNA can cause the destruction of ribosomes and death

of the cell; o  the "leaky" expression of T7 RNA polymerase can cause plasmid instability; o  it has been reported that, in the presence of IPTG, even an empty pET

plasmid is toxic for E.coli.

•  co-overexpression of the T7 lysozyme (that inhibits T7 RNA Pol) from compatible plasmids pLysS and pLysE;

•  T7 lysozyme hydrolyzes the peptidoglycan of the cell wall; •  insertion of the lac sequence downstream of the T7 promoter to reduce to the

minimum "leaky” transcription; •  selection of the better strains than the traditional BL21 (DE3) in countering

the effects of overexpression of toxic proteins (eg. cya mutants, cAMP deficient).


•  Promoter of gene 10 of phage T7 • 

pET protein expression system

•  Nuovi ceppi per l’espressione mediata dalla T7 RNApol •  BL21-AI (Invitrogen) copy of the T7 RNApol gene on chromosome under the

control of the promoter araBAD (strongly repressed by glucose). •  KRX (Promega) as above, but PrhaBAD promoter (repressed rhamnose).

•  T7 Express strains (New England Bioloabs) as above, wild-type lac promoter but controlled by the repressor lacIq, from LysY (without amidase activities) or by a combination of the two. These accessory genes are carried by a mini-F plasmid, stably maintained without use of antibiotics.

•  OverExpressTM C41(DE3) e C43(DE3) (Lucigen, Miroux and Walker 1996) isolated from BL21 (DE3), both have a mutation of GT to AA in the region -10 of the promoter T7 RNApol, such as to transform it from lacUV5 in wt lac.


NB: the CONTROL consists in the same strain under examination containing the plasmid without the insert (i.e. no recombinant protein), subjected to the same procedures and to the same conditions !

C41(DE3) C43(DE3)

(i ) Characteristics of the expression vector •  Nuovi ceppi per l’espressione mediata dalla T7 RNApol •  BL21-AI (Invitrogen) copy of the T7 RNApol gene on chromosome under the

control of the promoter araBAD (strongly repressed by glucose).

•  KRX (Promega) as above, but PrhaBAD promoter (repressed rhamnose). •  T7 Express strains (New England Bioloabs) as above, wild-type lac promoter but

controlled by the repressor lacIq, from LysY (without amidase activities) or by a combination of the two. These accessory genes are carried by a mini-F plasmid, stably maintained without use of antibiotics.

•  OverExpressTM C41(DE3) e C43(DE3) (Lucigen, Miroux and Walker 1996) isolated from BL21 (DE3), both have a mutation of GT to AA in the region -10 of the promoter T7 RNApol, such as to transform it from lacUV5 in wt lac.

•  Lemo21(DE3) (“Walker” strain) the activity is regulated by the T7 variant LysY whose expression can be modulated thanks to the promoter PrhaBAD (1-1000 mM rhamnose).

•  Translatability: - transcription start

In prokaryotes: - the signal is the RBS (ribosome binding site, or Shine- Dalgarno sequence)

mRNA

UAAGGAGG AUUCCUCC

AUG 5’ 3’

3’ 5’ 16S rRNA

small ribosomal subunit


- optimal distance from ATG = 8 nucleotides (4 ÷ 14)

the stronger the pairing between the Shine-Dalgarno sequence and 16S RNA, the more efficient translation initiation → the vector must be designed properly.

The downstream sequence immediately adjacent to the ATG must not contain regions of complementarity capable of forming hairpins.


•  Translatability: - transcription start

In prokaryotes: - the signal is the RBS (ribosome binding site, or Shine- Dalgarno sequence)

(ii ) Characteristics of the gene of interest

•  Translatability: - codon usage

•  Among the organisms that adopt the universal genetic code, the use the individual codons may be different:

- more codons encode the same amino acid - among these, some are used less (/more) often than others; - the corresponding tRNAs are under (/over) -represented in the cell.

•  Different organisms can use different codons with different frequencies.

AGA




•  It has been reported that the presence of rare codons in the heterologous overexpressed gene affects:

- the level of accumulation of the recombinant protein; - the mRNA stability of the plasmid; - in extreme cases it inhibits protein synthesis and bacterial growth.

- An important effect, although less obvious and evident, of codons rare (AGA) is the wrong introduction of Lys instead of Arg (misincorporation), especially when the cells are grown in minimal medium.

- Other effects: frame-shifting and termination of translation.



•  Ex: expression of different portions of a mitochondrial protein from S.cerevisiae in fusion with β-galactosidase


β-gal

Sc_mt-protein

Coomassie-stained gel immunoblot with anti-β-gal

GST

BL21-CodonPlus

-RP

BL21-CodonPlus

-RIL BL21-pLys

GST-Tyr

I NI I NI I NI I NI 97 66

45

30

20

-  site-specific mutagenesis to replace rare codons with favorite synonym codons for E.coli;

-  expression in engineered mutant strains (Rosetta) that express greater amounts of the required rare codons (by modification of the endogenous promoters or by introduction into the chromosome of clusters coming from phage genomes).



The question of the time required for folding is not trivial. If, for example, we consider a protein of 100 amino acids and it is assumed that each residue can take on three possible orientations in space, the structures that the polypeptide chain should try to find the correct conformation should be 3100, ie 5x1047. Even considering a very short time (10-13 seconds) for conversion from one structure to another one, the polypeptide would need to 1.6x1027 years to explore all the possible conformations, i.e. a much larger time than the age of the universe ( 2x1010 years).

In cells folding is a process assisted by chaperones.

(iii ) Characteristics of the recombinant protein •  Folding

•  The over-expression of heterologous proteins in E. coli often accompanies the inability to take the right structure; •  → segregation into insoluble aggregates, known as inclusion bodies.

•  Their formation depends on the rate of synthesis and the growth conditions; •  it is considered beneficial because it protects the protein from degradation and simplifies the purification, •  provided that the in vitro refolding is possible for the protein (solubilization, refolding, refolding vs. aggregation).

•  It is not obvious that the in vitro refolding permits to obtain the protein produced in good quantity and biologically active.


•  traditional strategy: reduction of the growth temperature (1). •  Alternative strategy: co-expression of proteins that assist the folding in vivo (2).

o  chaperones (DnaK-DnaJ-GrpE systems and GroEL-GroES) o  foldases (PPIasi, peptidyl prolyl cis / trans isomerases) o  promote the proper isomerization and targeting by transiently interacting with folding intermediates and accelerating the limiting steps of the folding process.

(1) Siller, E, et al. (2010) Slowing bacterial translation speed enhances eukaryotic protein folding efficiency. J Mol Biol 396: 1310-8 .

(2) Kolay, O, et al. (2009) Use of folding modulator to improve heterologous protein production in Escherichia coli. Microbial Cell Factories 8: 9.

(iii ) Characteristics of the recombinant protein •  Folding in the cytoplasm

•  Protein stability

•  Folding and proteolytic degradation are closely related •  Proteases, that are numerous. Among the most studied: - FTSH, ClpAP and ClpXP act specifically on proteins labeled by the "tag" AANDENYALAA, introduced through a mechanism that provides the modification of the 3 'of damaged mRNA molecules; - Lon ClpYQ and, more generally, act on truncated polypeptides.

→ Strains with mutated or knocked-out genes for proteases. Not devoid of disadvantages: - FTSH is an essential gene, only temperature-sensitive mutants can be used. It is a protease active even under denaturing conditions; - Lon inactivation produces filamentous cell aggregates; - (OmpT is adsorbed to the surface of the inclusion bodies during the purification and degrades the recombinant protein during the refolding phase)!

(iii ) Characteristics of the recombinant protein

- The half-life of a protein varies from a few hours to> 24 hours - Factors affecting stability: the amino acids to 'N-terminus of the recombinant protein affect the degradation rate of the protein - Presence of internal PEST sequences (rich in Pro, Glu, Ser, Thr) increase susceptibility to proteolytic degradation



fusion partner gene of interest

protease cleavage sequence

(iii ) Characteristics of both the recombinant protein and (i) the expression vector


•  Protection of the N-terminus of the protein to be expressed •  Originally introduced to facilitate the purification, •  also provided with a sequence useful for a specific proteolytic cleavage

MBP Fusion protein

Purified recombinant protein

Eluizione

Resina con legato maltosio

-

Sec translocase

Leader peptidase

Signal sequence

(iii ) Characteristics of both the recombinant protein and (iv) the strain used for expression

ABC transporter

adaptor protein

pore

•  the problem of post-translational modifications


•  How to choose the expression system: •  Which kind of protein are you interested in? When you want to express a protein of prokaryotic origin, the most obvious choice is E. coli. The method is fast and cheap, and the organism is fully equipped to perform the folding and post-transcriptional modifications required. In case the protein is of eukaryotic origin, the choice of the host organism and the method for obtaining expression will depend on many different factors (see further on). •  Is the protein synthesized in E.coli soluble? In some cases it is possible to re-solubilise the protein from inclusion bodies or increase its solubility by expressing the protein at low temperature. Even the expression of the protein of interest in fusion with a partner such as glutathione-S-transferase (GST) or maltose binding protein (MBP) can increase the solubility. Often, however, it is preferable to switch to a eukaryotic expression system, which of course is better equipped for the synthesis of proteins of eukaryotic origin. •  Which is the codon usage of the gene of interest? •  Does the preotein require post-translational modification for its function/activity? Steps forward have been made for the introduction some modification (e.g. for disulfide bridges see strains "Origami”)

http://wolfson.huji.ac.il/expression/index.html

for studying:

•  MOLECULAR BIOTECHNOLOGY Glick, Pasternak, Patten – 4th edition – chapter 6 •  Rosano & Ceccarelli (2014) Recombinant protein expression in

Escherichia coli: advances and challenges. Frontiers in Microbiology, 5, article 172

•  Baolei Jia & Che Ok Jeon (2016) High-throughput recombinant protein

expression in Escherichia coli: current status and future perspectives. Open Biology, 6: 160196

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