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 - PowerPoint PPT Presentation
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 . Introduction to Isotope Labeling of Proteins For NMR • 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
Transcript
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 .
Introduction to Isotope Labeling of Proteins For NMR
• 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
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 β-D-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 ExpressionT7 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
LB Broth Recipe (Luria-Bertani) 10 g tryptone 5 g of yeast extract 10 g of NaCl
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
Introduction to Isotope Labeling of Proteins For NMR
14 liter bioreactor
Introduction to Isotope Labeling of Proteins For NMR
Biotechnology Letters (1999) 12,1131
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 g 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
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 exchangec) Ni column d) heparine) 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
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 both 13C glucose and 15N NH4Cl can be added simultaneously
Journal of Biomolecular NMR, 20: 71–75, 2001.
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?
Introduction to Isotope Labeling of Proteins For NMR
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 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
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
J. Am. Chem. Soc. 1998, 120, 5591-5592
15N-labeled
Protein of Interest
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), 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 1H2H 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
Introduction to Isotope Labeling of Proteins For NMR
[3,3-2H2]-13C 2-ketobutyrate.
[2,3-2H2]-15N, 13C Val
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
