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!

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
 large collections of plasmids with unique
features and functions
- see:
http://www.the-scientist.com/yr1997/sept/profile2_970901.html
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
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)

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
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, don’t want to cleave plasmid multiple times
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 βD-thiogalactoside) to remove LacI repressor protein. IPTG binds LacI which no longer binds the
promoter region allowing transcription to occur

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.
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
LB Broth Recipe
(Luria-Bertani)
10 g tryptone
5 g of yeast extract
10 g of NaCl
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)

Cell growth in a Shake flask
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
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
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
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

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
 try different protein sequences

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”
 E. coli uses glucose and NH4Cl to synthesize all amino-acids  protein
 added prior to expressing protein of interest
13C glucose and 15N NH4Cl can be added simultaneously
 both
Journal of Biomolecular NMR, 20: 71–75, 2001.
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
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
13C and 15N labeled amino acid prior to protein expression
 Add
• 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
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
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
13C, 15N NMR resonances for labeled component of complex
 only “see”
 can see interactions (NOEs) between labeled and unlabeled compoents

J. OF BIOL. CHEM. (2003) 278(27), 25191–25206
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)
2
 level of H labeling depends on the
percent D2O the cells are grown in
 aromatic side-chains will be highly
protonated if 1H-glucose is used
2
1
 exchange labile N H to N H by
temperature increase or chemical
denaturation of the protein

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
[3,3-2H2]-13C 2-ketobutyrate.
[2,3-2H2]-15N, 13C Val
to the growth media.
1
1
 use H-glucose to generate H-aromatic side-chains
Metabolic pathway for generating
1H-methyl-Ile