3.3: Appendix I with Methods

General microbiological methods

Most laboratory strains of E. coli bacteria can grow in a defined culture medium consisting of a few inorganic compounds, trace amounts of several ions, an organic compound that can serve as an energy source as well as a source of carbon atoms, and one or more organic compounds required by the particular strain. This type of medium is referred to as “minimal medium”, containing only substances essential for growth. The growth of E. coli can be accelerated in a medium supplemented with amino acids, vitamins, pyrimidines and purines, in which cells do not have to expend time and energy synthesizing these substances. Culture media containing the added nutrients such as those derived from meat extract or yeast extract are referred to as “rich” media.

Growth of E. coli cells:

An E. coli culture can be propagated after transferring an inoculum of E. coli from a storage medium into a liquid medium. The cells first are in a period of nongrowth, called the lag phase during which the cells synthesize the components necessary for protein, DNA and RNA synthesis (Figure 1a). The E. coli cells then enter a phase of exponential growth called the log phase. The rate of growth of E. coli is dependent on the genotype of the strain, the type of medium, the temperature, the degree of aeration as well as the time the cells have been in stationary phase prior to growth. Exponential growth of E. coli continues until oxygen becomes limiting or the compostion of the medium changes. The E. coli cells then enter the stationary phase during which the cell number does not increase and the culture becomes saturated with cells. The growth of an E. coli culture can be monitored by spectrophotometry (A at 600 nm). The absorbance of a culture is proportional to the mass of cells in the sample.

Figure \(\PageIndex{Ia}\): Pattern of growth for a population of E. coli grown in liquid culture

Compound	Source	MW
Tris Trihydroxymethylaminomethane stored RT	Boehringer Mannheim	121
PEP (stable only at neutral pH) Phosphoenolpyruvic acid stored -20° salt: monosodium	Sigma	190
NADH (chemically unstable, store at neutral pH) Nicotinamide adenine dinucleotide disodium salt stored -20°	Sigma	709.4
EDTA (need to think about neutralization ) Ethylenediaminetetracetic acid disodium salt stored RT	Aldrich	372.24
NaCl Sodium chloride stored RT	Mallinckrodt	58.4
Imidazole stored RT	Aldrich	68.08
4Mg(OAc)2•4H2O Glycine - HCl (stored RT	Sigma Mallinckrodt	214.5 75
ATP (chemically unstable at acidic pH) Adenosine 5’-triphosphate, (disodium salt) stores -20°	Mallinckrodt	551.1
Potassum Hydroxide stored RT	Mallinckrodt	56
Acetic Acid stored RT	Mallinckrodt	60
SDS Sodium dodecylsulfate stored RT	Life Technologies	288.38
mercapto ethanol (smells) stored RT (use in hood)	Sigma	78.1
NH4Cl Ammonium chloride stored RT	Mallinckrodt	53.5
ribose 5-phosphate, disodium salt stored -20°	Sigma	274
pyruvate kinase ammonium sulfate suspension, store at 4°C	specific activity	447U/mg
lactic acid dehydrogenase, store at 4°	specific activity	720U/mg

 

General Methods for Protein Purification and Protein Determination

DNA Precipitation

One of the first steps in any protein purification is the removal of the nucleic acids. Rapidly proliferating cells contain a large amount of nucleic acids including DNA and ribosomal material. The DNA often makes the solutions very viscous. In general nucleic acids can be precipitated by forming complexes with polycationic material such as protamine sulfate. This material is difficult to dissolve and must be neutralized. All forms of DNA and m, t and rRNAs are precipitated using this method. The amount of protamine sulfate added to remove nucleic acid and not protein is determined by trial and error and is dependent on the source of the protamine sulfate. Polyethyleneimine (PEI) is a second polycationic macromolecule that also has been used to precipitate nucleic acis. A second method to reduce the viscosity of your solutions is to use the enzmyes DNAse and or RNAses which degrade DNA and RNA nonspecifically. Finally, streptomycin sulfate is a cationic natural product that interacts specifically with ribosomes that you will use to remove some of the nucleic acid. The amount of streptomycin sulfate that maximally precipitates nucleic acid and minimizes the precipitation of the protein of interest is determined by trial and error.

Determination of the Protein Concentration

There are many methods to determine the concentration of a protein. The method of choice is dependent on the amount of protein available and the presence of chemicals that could interfere with any one assay. The best way to determine the concentration is the use of the protein's \(\epsilon\). All proteins absorb light in the ultraviolet region at 280 nm. This contrasts with nucleic acids which also absorb light in the ultraviolet region, but predominantly at 260 nm. The predominant contributors to the protein absorption at 280 nm are tryptophan (W) and tyrosine (Y). There are now methods to calculate the \(\epsilon\) based on the proteins amino acid composition (Protein Science 4, 2411-2432 (1995), C. N. Pace, F. Vajdos, L. Fee, G. Grimsley and T. Gray). In general these method do pretty well when compared with the standard method of determining the proteins extinction coefficent.

We will use a colorimetric method and a protein standard bovine serum albumin (BSA) to determine our protein concentration. In general the choice of protein concentration is critical, as at high or low protein concentrations Beer's law is nonlinear. Use several concentrations of protein to ensure that that you have a concentration that is on the linear part of your standard curve.

All colorimetric assays have the problem that each protein reacts differently with the colorimetric reagent. The protein values obtained are thus relative values to some standard. Thus when reporting your protein concentrations, it is critical to report both the colorimetric method and the protein used as a standard. As noted above many reagents interfere with the standard assay. This interference can be be corrected for by running the standard curve.

We will use the Lowry reagent (O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall (1951) J. Biol Chem. 193, 265). This method can be used to detect 2 to 100 mg of protein. The method is based on the observation that Cu²⁺ under alkaline conditions forms a complex with peptide bonds of proteins and becomes reduced to Cu¹⁺. The side chains of the amino acids tyrosine, cysteine and tryptophan as well as Cu¹⁺ can react with Folin reagent which goes through a number of intermediates before eventually producing a blue color. Agents that acidify the solution such as strong acid or high concentrations of ammonium sulfate interfere with the assay as does EDTA which chelates copper or dithiothreitol or mercaptoethanol that cause the reduction of Cu²⁺. Thus, it is critical that you remove these complicating reagents before carrying out an assay.

Ultrafiltration

Centricon Ultrafiltration: The procedure is taken from the centriprep operating manual published by Amicon "Centiprep Filter Devices are disposable ultrafiltration devices used for concentration and desalting biological samples in 2 to 15 mL range.

Protein Molecular Weight Standards

References

General laboratory texts:

1. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press, New York.
2. Current Protocols in Molecular Biology (1992) (Eds. F.M. Ausubel et. al.). John Wiley & Sons, New York.
3. Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, New York.
4. Coupled Assays: Analytical Biochemistry 99, 142-145 (1979).
5. The Kinetics of Enzyme Catalyzed Reactions with two or more Substrates or Products: Nomenclature and Rate Equations W. W. Cleland Biochem. Biophys. Acta 67, 104-137 (1963).