Overview of Protein Expression

1
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Expression systems are based on the insertion of
  a gene into a host cell for its
• translation and expression into protein .

• Many recombinant proteins can be expressed to
  high
• levels in E. coli systems.
• most common choice for expressing labeled
• proteins for NMR
• Yeast (Pichia pastoris, Saccaromyces cerevisiae)
  is an
• alternative choice for NMR protein samples
• issues with glycosolyation of protein, which is
  not
• a problem with E. coli.
• choice between E. coli and yeast generally
  depend
• on personal experience.
• Insect cells (Baculovirus) and mammalian cell
  lines
• (CHO) are very popular expression systems that
  are
• currently not amenable for NMR samples
• no mechanism to incorporate isotope labeling or
• the process is cost prohibitive
• 15N labeling in CHO cells can cost 150-250K!

2
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• First step of the process involves the insertion
  of the DNA coding region of the protein
• of interest into a plasmid.
• plasmid - small, circular pieces of DNA that are
  found in E. coli and many other bacteria
• generally remain separate from the bacterial
  chromosome
• carry genes that can be expressed in the
  bacterium
• plasmids generally replicate and are passed on
  to daughter cells along with the
• chromosome
• Plasmids are highly infective, so many of the
  bacteria will take up the particles from
• simple exposure.
• Treating with calcium salts make membranes
  permeable and increase uptake of
• plasmids
• Plasmids used for cloning and expressing
  proteins are modified natural vectors
• more compact and efficient
• unnecessary elements removed
• Some Common plasmids
• pBR322
• pUC19
• pBAD

3
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Basic Features of a Plasmid

Defined region with restriction sites for
inserting the DNA
Gene that provides antibiotic resistance
(ampicillin resistance in this case)
replication is initiated
4
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Restriction Enzymes
• Recognizes and cuts DNA only at
• particular sequence of nucleotides
• blunt end cleaves both ends
• sticky ends cleaves only one strand
• Complimentary strand from DNA insert will
  match sticky end and insert in plasmid
• followed by ligation of the strands (T4 DNA
  Ligase)

5
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Restriction Enzymes
• Very large collection of restriction enzymes
  that target different DNA sequences

6
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Restriction Enzymes
• Restriction Map of plasmid showing the location
  where all restriction enzymes will
• cleave.
• allows determination of where how to insert a
  particular DNA sequence
• want a clean insertion point, dont want to
  cleave plasmid multiple times

7
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Next step of the process involves getting E.
  coli to express the protein from the plasmid.
• this occurs by the position of a promoter next
  to the inserted gene
• two common promoters are
• lac complex promoter
• T7 promoter

lac complex promoter Transcription is simply
switched on by the addition of IPTG (isopropyl
ß-D-thiogalactoside) to remove LacI repressor
protein. IPTG binds LacI which no longer binds
the promoter region allowing transcription to
occur
8
Introduction to Isotope Labeling of Proteins For
NMR
Overview of Protein Expression

• T7 promoter Again, transcription is switched on
  by the addition of IPTG to remove LacI repressor
  protein.
• IPTG binds LacI which no longer binds the
  promoter region allowing transcription/production
  of T7 RNA polymerase to occur.
• T7 RNA polymerase binds the T7 promoter in the
  plasmid to initiate expression of the protein
• two-step process leads to an amplification of
  the amount of gene product - produce very high
  quantities of protein.

9
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Next step of the process involves growing the E.
  coli cells
• Shake Flask
• cells are place in a growth media that
  provides the
• required nutrients to the cell
• amino acids, vitamins, growth factors, etc
• shake the flask at a constant temperature of 37O
• keeps homogenous mixture
• increases oxygen uptake
• grow cells to proper density (OD 0.7 at 600nm)

LB Broth Recipe (Luria-Bertani) 10
g tryptone 5 g of yeast extract 10 g of NaCl
Cell growth in a Shake flask
10
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Next step of the process involves growing the E.
  coli cells
• Bioreactors
• more efficient
• higher production volumes
• can be 100s of liters in size
• Can grow cells to a higher density
• better control of pH
• better control of oxygen levels
• better control of temperature
• better control of mixing
• sterile conditions

14 liter bioreactor
11
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Next step is to harvest and lysis the cells and
  purify the protein
• Now that E. coli is producing the desired
  protein, need to extract the protein from the
  cell and purify it.
• the amount of protein that can be obtained from
  an expression system is highly variable and can
  range from mg to mg to even g quantities.
• it depends on the behavior of the protein,
  expression level, method of fermentation and the
  amount of cells grown

over-expressed protein
Biotechnology Letters (1999) 12,1131
12
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Cell Lysis
• A number of ways to lysis or break open a cell
• Gentle Methods
• Osmotic suspend cells in high salt
• Freeze-thaw rapidly freeze cells in liquid
  nitrogen and thaw
• Detergent detergents (DSD) solubilize cellular
  membranes
• Enzymatic enzymatic removal of the cell wall
  with lysozyme
• Vigorous Methods
• Sonication sonicator lyse cells through shear
  forces
• French press cells are lysed by shear forces
  resulting from forcing cell
• suspension through a
  small orifice under high pressure.
• Grinding hand grinding with a mortar and
  pestal
• Mechanical homogenization - Blenders or other
  motorized devices to grind
  cells
• Glass bead homogenization - abrasive actions of
  the vortexed beads break
• 
  cell walls

French Press
13
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Protein Purification - A large number of ways to
  purify a protein
• protocols are dependent on the protein
• chromatography is a common component of the
  purification
• protocol where typically multiple columns are
  used
• a) size-exclusion b) ion exchange
• c) Ni column d) heparin
• e) reverse-phase f) affinity column
• dialysis for buffer exchange and removal of
  low-molecular weigh
• impurities
• To increase the ease of purifying a protein
  generally include a unique
• tag sequence at the N- or C-terminus
• HIS tag add 6 histidines to the N- or C-
  terminus
• - preferentially binds Ni column
• FLAG tag DYKDDDDK added to terminus
• - preferentially binds M1 monoclonal antibody
  affinity
• column
• glutathione S-transferase (GST) tags fusion
  protein
• - readily purified with glutathione-coupled
  column

14
Introduction to Isotope Labeling of Proteins For
NMR

• Overview of Protein Expression
• Some Common Problems
• protein is not soluble and included in inclusion
  bodies
• insoluble aggregates of mis-folded proteins
• inclusion bodies are easily purified and can be
  solubilized using denaturing conditions
• How to re-fold the Protein?
• Finding a re-folding protocol may take
  significant effort (months-years?) and
• involve numerous steps
• something to be avoided if possible
• protein is toxic to cell
• find a different expression vector or use a
  similar protein from a different
• organisim
• proper protein fold
• proper disulphide bond formation may need to
  re-fold the protein
• presence of tag may inhibit proper folding may
  need to remove the tag
• low expression levels
• try different plasmid constructs

15
Introduction to Isotope Labeling of Proteins For
NMR

• 13C and 15N Isotope Labeling of the protein
• cells need to be grown in minimal media
• use 13C glucose to achieve 100 uniformed 13C
  labeling of protein
• use 15N NH4Cl to achieve 100 uniformed 15N
  labeling of protein
• glucose and NH4Cl are sole source of carbon and
  nitrogen in minimal media

Journal of Biomolecular NMR, 20 7175, 2001.
16
Introduction to Isotope Labeling of Proteins For
NMR

• 13C and 15N Isotope Labeling of the protein
• Usually isotope labeling does not negatively
  impact protein expression
• Some Common Problems with Isotope Labeling
  Problems
• minimal media stresses cells
• slower growth
• typically lower expression levels
• isotope labeling of All proteins
• minimal isotope affect may affect
• enzyme activities
• isotope labeling of expressed protein may
• affect protein properties
• solubility?
• proper folding?

1H-15N HSQC spectra of 13C,15N labeled protein
17
Introduction to Isotope Labeling of Proteins For
NMR

• 13C and 15N Isotope Labeling of the protein
• Can introduce specific amino acid labels
• A variety of 13C and 15N labeled amino acids are
  commercial available
• Add saturating amounts of 19 of 20 amino acids
  to minimal growth media
• Add 13C and 15N labeled amino acid prior to
  protein expression
• media is actually very rich and the cells grow
  very well
• cells exclusively use the supplied amino-acids
  to synthesize proteins
• all of the occurrences of the amino-acid are
  labeled in the protein
• may be some additional labeled residues if the
  labeled amino
• acid is a precursor in the synthesis of other
  amino acids.

1H-15N-HSQC of His, Tyr Gly labeled SH2-Domain
no mechanism to label one specific amino acid i.e
Gly-87
18
Introduction to Isotope Labeling of Proteins For
NMR

• 13C and 15N Isotope Labeling of the protein
• Can label specific segment in protein
• use peptide splicing element intein (Protozyme)
• inteins are insertion sequences which are
  cleaved off after translations
• preceding and succeeding fragments are ligated ?
  extein

Protein of Interest
15N-labeled
J. Am. Chem. Soc. 1998, 120, 5591-5592
19
Introduction to Isotope Labeling of Proteins For
NMR

• 13C and 15N Isotope Labeling of the protein
• Can also label only one component of a complex
• simply mix unlabeled and labeled components to
  form the complex
• greatly simplifies the NMR spectra
• only see 13C, 15N NMR resonances for labeled
  component of complex
• can see interactions (NOEs) between labeled and
  unlabeled compoents

J. OF BIOL. CHEM. (2003) 278(27), 2519125206
20
Introduction to Isotope Labeling of Proteins For
NMR

• 2H Labeling of the protein
• simply requires growing the cells in D2O
• severe isotope effect for 1H?2H
• stresses the cell
• E. coli needs to be acclimated to
• D2O
• pass cells into increasing
• percentage of D2O
• cell growth slows significantly in
• D2O (18-60 hrs)
• level of 2H labeling depends on the
• percent D2O the cells are grown in
• aromatic side-chains will be highly
• protonated if 1H-glucose is used
• exchange labile N2H to N1H by
• temperature increase or chemical
• denaturation of the protein

21
Introduction to Isotope Labeling of Proteins For
NMR

• 2H Labeling of the protein
• As we have seen, deuterium labeling a protein
  removes a majority of protons necessary for
  protein structure calculation
• can introduce site specific protonation to
  regain some proton based distance
• constraints
• label the methyl groups of Leu, Ile, and Val by
  adding
• to the growth media.
• use 1H-glucose to generate 1H-aromatic
  side-chains

3,3-2H2-13C 2-ketobutyrate.
2,3-2H2-15N, 13C Val
Metabolic pathway for generating 1H-methyl-Ile