1. 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  15 N 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  large collections of plasmids with unique features and functions - see: http://www.the-scientist.com/yr1997/sept/profile2_970901.html

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, don’t 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 β- 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 37 O – keeps homogenous mixture – increases oxygen uptake  grow cells to proper density (OD ~ 0.7 at 600nm) Cell growth in a Shake flask LB Broth Recipe (Luria-Bertani) 10 g tryptone 5 g of yeast extract 10 g of NaCl

10. 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

11. 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

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 columnd) heparin e) reverse-phasef) 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  try different protein sequences

15. Introduction to Isotope Labeling of Proteins For NMR 13 C and 15 N Isotope Labeling of the protein cells need to be grown in “minimal media” use 13 C glucose to achieve ~ 100% uniformed 13 C labeling of protein use 15 N NH 4 Cl to achieve ~ 100% uniformed 15 N labeling of protein  glucose and NH 4 Cl 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 13 C glucose and 15 N NH4Cl can be added simultaneously Journal of Biomolecular NMR, 20: 71–75, 2001.

16. 13 C and 15 N 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 1 H- 15 N HSQC spectra of 13 C, 15 N labeled protein

17. Introduction to Isotope Labeling of Proteins For NMR 13 C and 15 N Isotope Labeling of the protein Can introduce specific amino acid labels A variety of 13 C and 15 N labeled amino acids are commercial available  Add saturating amounts of 19 of 20 amino acids to minimal growth media  Add 13 C and 15 N 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. 1 H- 15 N-HSQC of His, Tyr & Gly labeled SH 2 -Domain no mechanism to label one specific amino acid i.e Gly-87

18. Introduction to Isotope Labeling of Proteins For NMR 13 C and 15 N 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 15 N-labeled Protein of Interest

19. Introduction to Isotope Labeling of Proteins For NMR 13 C and 15 N 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” 13 C, 15 N 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

20. Introduction to Isotope Labeling of Proteins For NMR 2 H Labeling of the protein simply requires growing the cells in D 2 O  severe isotope effect for 1 H  2 H stresses the cell  E. coli needs to be acclimated to D 2 O  pass cells into increasing percentage of D 2 O  cell growth slows significantly in D 2 O (18-60 hrs)  level of 2 H labeling depends on the percent D 2 O the cells are grown in  aromatic side-chains will be highly protonated if 1 H-glucose is used  exchange labile N 2 H to N 1 H by temperature increase or chemical denaturation of the protein

21. Introduction to Isotope Labeling of Proteins For NMR [3,3- 2 H 2 ]- 13 C 2-ketobutyrate. [2,3- 2 H 2 ]- 15 N, 13 C Val 2 H 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 1 H-glucose to generate 1 H-aromatic side-chains Metabolic pathway for generating 1 H-methyl-Ile