Concepts in Chemical Biology

8/12/2019 Concepts in Chemical Biology

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Lecture 4

Recombinant protein expression,purification, and cell culture

CHEM 570, Fall 2013Concepts in Chemical Biology

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Prokaryotic cells arelaboratory workhorses

for recombinantprotein expression

protein builds upin cytoplasm

Prokaryotic cells arelaboratory workhorses

for recombinantprotein expression


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4-3Overview of protein expression

4-4Restriction enzymesCut the phosphate backbone of double-stranded DNA

Sequence-specific cleavage of DNA:

Sticky ends:

GAATTC

CTTAAG

G

CTTAA

AATTC

G+EcoRI

Blunt ends:

CCCGGG

GGGCCC

CCC

GGG

GGG

CCC+

SmaI

Most restriction sites are palindromic .

Nobel Prize in Physiologyor Medicine (1978)

to Arber, Nathans, and Smith


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4-7PCR for site-directed mutagenesis

There are many PCR-based methods for mutagenesis

This particular method is often called QuikChange (Stratagene)

Nobel Prize inChemistry (1993)to Michael Smith

4-8Elements of recombinant expression

Plasmid origin of replication ( ori )Copy number; high copy number (>20 per cell)leads to larger amounts of protein

Antibiotic resistance marker ampicillin, kanamycin, chloramphenicol,tetracycline...

Inducible promoter IPTG

In 2003, ~80% of proteins used to obtain 3D structureswere made via recombinant expression in E. coli

J. Biotechnol. 2006 , 115 , 113


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transcription

translation

ORF (gene)RBSpromoter terminator

5 3DNA

RNA

protein

Elements of recombinant expression

4-10Elements of recombinant expressionmessenger RNA (mRNA)

Translation initiation region: ribosome binding site (RBS)Shine-Dalgarno sequence, ~7 nt from AUGAGGAGGU in E. coli (see Slide 2-42)

Translation initiation codonAUG start codon (Met)

Transcriptional terminator Downstream of STOP codon, stem-loop at 3-end

Translational terminators: STOP codonsUAA (ochre), UAG (amber **), and UGA (opal)UAA is preferred in E. coli

** Discovered by graduate student Harris Bernstein;Bernstein means amber in German ( Genetics 1995 , 141 , 439)


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4-13Using T7 systems for expressionFor high expression levels of recombinant proteins

Utilizes bacteriophage T7 promoter 20 nt sequence not recognized by E. coli RNA polymerase Strong promoter leads to elevated transcription T7 RNA pol 230 nt/s; E. coli RNA pol 50 nt/s Normal strains of E. coli do not synthesize T7 RNA polymerase Must use a strain of E. coli that produces this enzyme

DE3 phage fragment contains gene for T7 RNA pol T7 RNA polymerase gene is on E. coli chromosome Inducible by IPTG

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Recombinant protein purificationRecombinant proteins are typically expressed

as a fusion with an affinity tag

Tag Protein of interest

Maltose Binding Protein (MBP) 40 kDa Amylose

Glutathione S-transferase (GST) 26 kDa Glutathione

Poly-His (His 6)


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Protease cleavage sites

Factor Xa: Ile-Glu-Gly-ArgX

Enterokinase: Asp-Asp-Asp-Asp-LysX

Thrombin: Leu-Val-Pro-ArgGly-Ser

Tobacco Etch Virus (TEV): Glu-Asn-Leu-Tyr-Phe-GlnGly

4-18Expression problemsCleavage of target protein by E. coli proteases

SOLUTION: engineered E. coli strains

The major E. coli proteasesare the Lon and OmpT proteases

Standard E. coli strains are lon and ompT minus


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Recall... 64 triplet DNA codons code for 20 aa (and 3 STOP )

Expression problemsDifferent codon usage between E. coli and mammalian genes

E. coli H. sapiens

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If a eukaryotic gene uses a codon that is rare for E. coli then the protein product will not be produced at a high level, or at all

SOLUTION: E. coli strains that have enhanced expressionof the AUA, AGA, AGG, CGG, CUA, CCC, and GGA tRNAs

tRNA population reflects the bias of the mRNA population

E. coli transformedwith pRARE tocreate Rosetta

strain

Expression problems

http://wolfson.huji.ac.il/expression/rosetta.pdf


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Aggregation of recombinant proteins High concentrations of folding intermediates Inefficient processing by chaperones

Aggregated proteins = inclusion bodies= insoluble aggregates

(misfolded and thus biologically inactive)

Expression problems

Without re-engineering of target

WITH re-engineering of target

What to do??

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Grow bacteria at reduced temperatures Aggregation is disfavored E. coli chaperones are more active at 30 C

Expression solutions

Re-engineer the target protein Fusion proteins Many fusion partners lead to high solubility

(MBP, GST, NusA...) But, of course, now you have something stuck

onto your protein

Screen for soluble variants (mutants)


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4-23Expression solutions

Top Ten Tips for Producing 13 C 15 N Proteins in Abundance:http://www.isotope.com/uploads/file/cilapp15.pdf (from Chad Rienstras lab)

Choose a different host strain

Elevated expression of F1F0-ATP synthase- C43(DE3) Avidis

Enabling disulfide bond formation- trxB-negative strains Novagen- lack thioredoxin reductase

Overexpression of chaperone proteins

Choose a different host organism Bacillus strains secrete proteins directly to

extracellular medium Good for high protein expression (up to 2 g/L!) Good for expressing toxic proteins

4-24Limitations of bacterial expression

If PTMs (phosphorylation, glycosylation, etc.)are critical for biological activity,

then need to choose another expression system

Post-translational modifications will not be performedin the bacterial host.

Only the standard 20 ribosomally encoded amino acidscan be incorporated

See upcoming Lecture 12 for unnatural amino acid incorporation


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4-25Expression in eukaryotic hosts

Appl. Microbiol. Biotechnol. 2004 , 65 , 363

Yeast (fungi) Secrete proteins Allow introduction of PTMs Protein yields up to 15 g/L Can be rather slow growth

Insect cells Closer resemblance to

mammalian cells than yeast

Human cells Expensive Low protein yields (


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4-27How to culture cancer cellsPrimary cells : cultured directly from animal/person

Most primary cell cultures have limited lifespan Senescence : cells viable, but stop dividing

Immortalized cells : ability to proliferate indefinitely Through random mutation Through deliberate modification (e.g., telomerase

expression) Numerous well-established immortalized cell lines

4-28How to culture cancer cellsSuspension culture : cells growing unattached to surface

(e.g., blood cells)

Adherent culture : cells growing on tissue culture plastic(e.g., most solid tissues)

Cell density matters! At high cell density... nutrients are depleted dead cells accumulate cell-cell contact growth arrest or differentiation

Steriletechnique iscritical uselaminar flow

hood


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4-29How to culture cancer cellsMedia changes

centrifugation (for suspension cultures) aspiration (for adherent cultures)

Passaging cells (splitting) small amount of cells transferred to new flask for adherent cultures, cells must first be detached

(trypsin or other enzymes)

Cell line contaminationis a big problem!

~20% of human cell lines arethought to be contaminated,often with fast-growing HeLa cells.

4-30Common cell linesHeLa cells

human cervical cancer cells from He nrietta La cks, age 31,

who died of cancer in 1951 Lacks did not know about or

give consent to use of her cells rapidly growing, 82 chromosomes, active telomerase

human papillomavirus (HPV) integrated into genome

CHO cells from Chinese h amster o varies


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4-31Common cell linesHEK 293 cells (aka HEK, or 293)

h uman e mbryonic kidney cells (epithelial) generated by transformation of normal human

embryonic kidney cells with adenovirus 5 DNA

HEK 293T cells variant of HEK 293 that contains the SV40 large T

antigen, which allows replication of plasmids withSV40 origin of replication

4-32Common cell lines3T3 cells

mouse embryonic fibroblast cells will stop growing at low densities does NOT form tumors in mice abnormal cells; multiple chromosome alterations one of first cells lines used to distinguish ability to grow

indefinitely ( immortalization ) and ability to form tumors

(oncogenic transformation )http://www.bioinfo.org.cn/book/Great%20Experments/great22.htm

Telomere shortening is a major cause of finite cell culture time.

Those cells lines that overcome telomere shorteningcan be immortal.

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