Tải bản đầy đủ - 0 (trang)
23 Preparations of S-30 Extract for Protein Synthesis In Vitro (Roberts and Paterson 1973)

23 Preparations of S-30 Extract for Protein Synthesis In Vitro (Roberts and Paterson 1973)

Tải bản đầy đủ - 0trang

140



7



Qualitative and Quantitative Estimations of Amino Acids and Proteins



6. Collect the eluant in fraction. Combine the most turbid fractions. Immediately,

dilute 20 mL of the combined fraction to 2 mL with water and measure the

absorbance. The A260/A280 should be above 1.6.

7. Freeze immediately and store the combined fractions in small aliquots

(0.2–0.5 mL) in microfuge tubes under liquid nitrogen (À190 C) or at À70 C.

Note

1. Embryos can be collected from freshly harvested wheat grains. Final purification

of embryos is carried out briefly by floating in a mixture of organic solvents –

cyclohexane: carbon tetrachloride (1:4) – change the solvent ratio slightly to

float the embryos on the surface. Collect the floating embryos quickly and dry on

filter paper. Embryos can be stored in sealed vials in freezers for a few weeks

before extraction.

2. The translation efficiency of the extract will be lower if A260/A280 is below 1.6.

Carefully combine only peak turbid fractions.

3. Storing of the extract at À20 C leads up to 70% activity loss in the 3 weeks.

4. The thawed extract should be used once.



7.24



In Vitro Translation Assay (Marcus et al. 1974)



In vitro translation study is an excellent procedure to study protein synthesis and to

characterize the product encoded by mRNAs. The cell-free protein synthesizing

system efficiently translates mRNAs from exogenous source under optimal

conditions. The assay is usually carried out in a small volume of 20–25 mL,

which could be scaled-up for preparative purposes.

Principle

The cell-free (S-30) extract containing the necessary protein synthesis machinery

components translates the genetic message in the mRNAs into protein when

provided with energy source under proper ionic conditions. The hot TCA perceptible radioactivity due to the labelled amino acid incorporation is measured.

Materials

1. Cell-free extract (see preceding experiment)

2. Salt mix (10Â)

200 mM HEPES-KOH (pH 7.6)

750 mM Potassium chloride

25 mM Magnesium acetate

20 mM Dithiothreitol

6 mM Spermidine (optional)

Store in aliquots at À20 C



7.24



In Vitro Translation Assay



141



3. Energy mix (5Â)

2.5 mM ATP (pH 7.0)

25 mL 10 mM ATP (dipotassium salt)

1.5 mM GTP(pH 7.0)

15 mL 10 mM GTP (trisodium salt)

100 mM creatine phosphate (pH 7.0)

10 mL 1 M creatine phosphate (dipotassium salt)

250 mg/mL creatine phosphokinase

25 mL creatine phosphokinase 1 mg/mL

25 mL H2O

Prepare the 5Â mix afresh from stock solution kept at À20 C.

4. Amino acid mix

2.4 mM each amino acid except the labelled amino acid stored at À20 C.

Procedure

1. In an Eppendorf tube mix successively:

10 mL of 10Â salt mix.

20 mL of 5Â energy mix.

10 mL of amino acid mix.

4 mL (>40 Ci) of 35S methionine (>1,000 Ci/immol) or 3H Leucine 36 mL of

S-30 extract (freshly thawn)

2. Pipette out into three numbered 0.5-mL Eppendorf tubes three different

volumes (1, 2 and 4 mL) of mRNAa (1 mg/mL) solution. Normalize the volume

to 5 mL in each tube by adding sterile distilled water.

3. Transfer 20 mL of the assay mix (step 1) to each tube containing mRNA.

4. To the remaining assay mixture add 5 mL of sterile water (control).

5. Mix and incubate all the tubes at 25 C for 1 h.

6. Meanwhile warm 5 mM square filter papers (Whatman 3 mm) over a hot plate

at 70 C.

7. After incubation transfer 2 or 5 mL of assay in triplicate onto the filters papers,

and dry.

8. Precipitate the proteins onto the filters by transferring them to ice-cold 10%

trichloro acetic acid (TCA) containing excess unlabelled amino acid for

10 min.

9. Boil the filters in 5% TCA for 10 min in a water bath to deacylate the charged

tRNAs.

10. Wash the filters successively in 5% TCA, ethanol, ethanol-ether mixture and

finally ether each step proceeding for 2–3 min.

11. Dry the filters at 70 C. Transfer each filter to a scintillation vial, add 2 mL

scintillation fluid (4 g PPO/L toluene) or suitable mixture and count the

radioactivity.



142



7



Qualitative and Quantitative Estimations of Amino Acids and Proteins



12. Include three filters with no solution pipetted onto them during the above

processing to subtract the background noise.

13. Arrest the reaction after incubation (step 5) by adding 5 mL of sample buffer

(5Â) for SDS-PAGE analysis.



7.25



Ammonium Sulphate Fractionation of Proteins



The solubility of proteins is markedly affected by the ionic strength of the medium.

As the ionic strength is raised, protein solubility at first increases, which is referred

to as “salting in”. However, beyond a certain point the solubility begins to decrease

and this is known as “salting out”.

As low ionic strengths the activity coefficients of the ionizable groups of the

proteins are decreased so that their effective concentration is decreased. This is

because surrounded by counter ions which prevent interaction between the ionizable

groups. Thus protein–protein interactions are decreased and the solubility is increased.

At high ionic strengths water becomes bound by the added ions which is not

enough to properly hydrate the proteins. As a result, protein–protein interactions

exceed protein–water interactions and the solubility decreases. Because of

differences in structure and amino acid sequence, proteins differ in their slating in

and salting out behaviour. This forms the basis for the fractional precipitation by

application of salt.

Ammonium sulphate is preferably a useful salt for the fractional precipitation of

proteins. It is available in highly purified form and has great solubility allowing

significant changes in the ionic strength. Moreover, it is not so expensive. Changes

in the ammonium sulphate concentration of a solution can be brought about either

by adding solid substance or by adding a solution of known saturation, generally,

a fully saturated (100%) solution (Table 7.5).



7.26



Methods for Determining Amino Acid Sequences

of Protein



The sequence of amino acids in proteins can be determined by means of three basic

analytical procedures:

(a) Identification of the NH2 – terminal amino acid in the protein.

(b) Identification of the COOH – terminal amino acid.

(c) Partial cleavage of the original polypeptide into smaller polypeptides whose

sequence can be determined.

In the last procedure, cleavage of the original protein must be carried out in at

least two different ways so that the smaller polypeptides produced in one procedure

“overlap” those produced in the second procedure and provide on opportunity for



0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100



45



50



g solid ammonium sulphate to add to 100 mL of solution

10.7

13.6

16.6

19.7

22.9

26.2

29.5

8.0

10.9

13.9

16.8

20.0

23.2

26.6

5.4

8.2

11.1

14.1

17.1

20.3

23.6

2.6

5.5

8.3

11.3

14.3

17.4

20.7

0

2.7

5.6

8.4

11.5

14.5

17.7

0

2.7

5.7

8.5

11.7

14.8

0

2.8

5.7

8.7

11.9

0

2.8

5.8

8.8

0

2.9

5.9

0

2.7

0



20

25

30

35

40

Initial concentration of ammonium sulphate

33.1

30.0

27.0

24.0

21.0

18.2

15.0

12.0

9.0

3.0

3.0

0



55



36.6

33.6

30.5

27.5

24.4

251.4

18.4

15.3

12.2

9.1

6.1

3.0

0



60



40.4

37.3

34.0

31.0

28.0

24.8

21.7

18.7

15.5

12.5

9.3

6.2

3.1

0



65



44.2

41.1

37.9

64.8

31.6

28.4

25.3

22.1

19.0

15.8

12.7

9.4

6.3

3.1

0



70



48.3

45.0

41.8

38.6

35.4

32.1

28.9

25.8

22.5

19.3

16.1

12.9

9.6

6.4

3.2

0



75



52.3

49.1

45.8

42.6

39.2

36.0

32.8

29.6

26.2

22.9

19.7

16.3

13.1

9.8

6.6

3.2

0



80



Table 7.5 The amount of solid ammonium sulphate to be added to a solution to give the desired final saturation at 0 C

Final concentration of ammonium sulphate, % saturation 0 C



56.7

53.3

50.0

46.6

43.3

40.1

36.7

33.4

30.0

26.7

23.3

20.0

16.6

13.4

10.0

6.7

3.3

0



85



61.1

57.8

54.5

51.0

47.6

44.2

40.8

37.4

34.0

30.6

27.2

23.8

20.4

17.0

13.6

10.2

6.8

3.4

0



90



65.9

62.4

58.9

55.5

51.9

48.5

45.1

41.6

38.1

36.7

31.2

27.7

24.2

20.8

17.3

13.9

10.4

6.9

3.4

0



95



70.7

67.1

63.6

60.0

56.5

52.9

49.5

45.9

42.4

38.8

35.3

31.7

28.3

24.7

21.2

17.6

14.4

10.6

7.1

3.5



100



7.26

Methods for Determining Amino Acid Sequences of Protein

143



144



7



Qualitative and Quantitative Estimations of Amino Acids and Proteins



identifying the sequence of amino acids in the area of the original chain where the

cleavage occurs. The protein whose structure is to be determined must obviously be

free of any contaminating amino acids or peptides. Knowing its molecular weight

and amino acid composition, the number of times each residue occurs in the protein

can be determined. The determination of sequence can proceed in following steps.



7.26.1



Identification of the NH2-Terminal Amino Acid



When a polypeptide is reacted with 2,4-dinitrofluorobenzene the NH2-terminal

group (and the e-amino group of any lysine that is present in peptide linkage) reacts

to form the intensely yellow 2,4-dinitrophenyl derivative of the polypeptide.

Subsequent hydrolysis of the peptide with 6 N HCl hydrolyzes all the peptide

bonds, and the yellow derivative of the NH2-terminal residue (and that of lysine)

can be separated by paper chromatography from the free amino acids, compared

with know derivatives of the amino acids, and identified. The NH2-terminal residue

can also be identified with the dansyl reagent.

The reaction of polypeptides with phenylisothiocyanate in dilute alkali is the

basis for a sequential degradation of a polypeptide that has been devised by

P. Edman. In this procedure, the NH2-terminal group reacts to form a phenylthicarbamyl derivate. Next, treatment with mild acid causes cyclization and cleavage

of the NH2-terminal amino acid as its phenylthiohydatoin. This compound can be

separated and compared with the same derivative of known amino acids and

thereby identified. The acid conditions utilized to cleave off the phenylthiohydantoin are not sufficiently drastic as to break any other peptide linkages. As a

consequence, this method results in the removal and identification of the

NH2-terminal amino acid together with the production of a polypeptide containing

one less amino acid than the original. This new polypeptide can now be treated with

more phenylisothiocyanate in alkali in the same manner and the process repeated

many times to degrade the original polypeptide in a stepwise manner (Fig. 7.1).



7.26.2



Identification of the COOH-Terminal Amino Acid



The carboxyl-terminal group of a polypeptide (and the distal carboxyl groups of

aspartic and glutamic acid residues in the peptide) can be reduced to the

corresponding alcohol with lithium borohydride, LiBH4. It is first necessary to

protect the free amino groups by acetylation and to esterify the carboxyl groups.

The polypeptide can then be hydrolyzed with acid to produce its constituent amino

acids and the amino alcohol corresponding to the COOH-terminal residue. The

alcohol can be separated, compared with reference compounds, and identified.

The action of the enzyme carboxypeptidase on polypeptides can also be used to

identify the COOH-terminal amino acid, since its action is to hydrolyze that amino



7.26



Methods for Determining Amino Acid Sequences of Protein



145



Phenylthiohydantoin

derivative of

NH2-terminal amino acid

Phenylisothiocyan ate



O



N

C

Alkali

R1

O



R



C



O



HN

R2



R

O



O



HC R4



HC

O



C

NH



R3



CH

C



R2



HC



NH

3



HN



Polypeptide



O



C



HN



C



+



HC

O



CH

C



R1



HN

2



NH

R



NH



CH

C



HN



3



C



HN



C



O



Anhydrous

acid



C



CH



HC



S



C



NH



S

+

NH2

R1



N

C



O



CH

C



O



HN

R4



C



Phenylthiocarbamyl derivative



HC

O



R4



C



Polypeptide lacking

NH2 -terminal amino acid



Fig. 7.1 Edman degradation for N-terminal amino acid



acid off the polypeptide. The major disadvantage is that the enzyme does not act

exclusively on the original polypeptide but will also hydrolyze the new COOHterminal peptide bond as soon as it is formed. Therefore, the investigator must

follow the rate of formation of free amino acids to learn which residue represents

the terminal in the original polypeptide.



146



7



7.26.3



Qualitative and Quantitative Estimations of Amino Acids and Proteins



Cleavage of Protein into Smaller Units



Both enzymatic and chemical procedures have been utilized to produce smaller

polypeptides that overlap in sequences with the present proteins. Partial hydrolysis

by dilute acid can be employed. Cyanogens bromide (CNBr) is also used since

conditions can be chosen which will cleave only those peptide bonds in which the

carbonyl group belongs to a methionine residue. The methionine residue becomes a

substituted lactone of homoserine that is bound to one of the two peptides produced

in the reaction. This procedure allows one to determine the amino acids in the

region of the methionine residues in the original peptide. In addition, knowing

the number of methionine residues in the original polypeptide, one can predict the

number of smaller polypeptides that will result from treatment with CNBr

(Fig. 7.2).

Proteolytic enzymes have been extensively used to cleave proteins into smaller

polypeptides which can then be analysed by the procedures described above. For

example, trypsin hydrolyze those peptide bonds in which the carbonyl group is

contributed by either lysine or arginine. As with the CNBr reaction, one can predict

the number of polypeptides that will be formed by the action of trypsin if the

number of lysine and arginine residues in the protein is known.

Chymotrypsin will hydrolyze those peptide bonds in which the carboxyl group

belongs to phenylalanine, tyrosine or typtophan. Pepsin cleaves the peptide bonds

in which the amino group is furnished by phenylalanine, typrosine, tryptophan,

lysine, glutamic and aspartic acids. By utilizing trypsin, whose action is quite



NH

R1



NH

R1



CH

C



O



New polypeptide containing

modified methoinine residue



CO



HN

HC



CH



HN

CH2



CH2 S



CH3 + CNBr



HC



CH2 + H3C



S



CN + HBr



CH2

O



O



C



R2



CH

C



O



NH2



NH

R2



C

+



O



Original polypeptide



Fig. 7.2 C-terminal sequencing of protein



CH

C



New polypeptide

produced on cleavage

O



7.26



Methods for Determining Amino Acid Sequences of Protein



147



specific, and either chymotrypsin or pepsin, the investigator can obtain fragments of

the original protein or polypeptide that overlap in sequence. Once the sequence of

amino acids in these fragments is known, the process of fitting together the

individual fragments can proceed. If, as in the case of insulin, the original protein

can be easily separated into two parts by simple reduction of disulfide bonds, the

sequence determination of the two separate chains can proceed.



Chapter 8



Protein Purification Techniques



A wide variety of protein purification methods that can be combined to generate a

suitable purification scheme are available. To understand the nature of a biomolecule like protein, it must be purified to near homogeneity. Purified protein may be

used for a cloning or may be used to learn about its catalytic activities and its

responsiveness to regulatory molecules that raise or lower the activity or

interactions with other proteins. For attaining the goal of a pure protein, the cardinal

rule is that the ratio of the target protein (in terms of its activity) to the total protein

is increased to the limit. Usually, one executes a series of purification steps.

Combining early ones of high capacity and low resolution (when large amounts

of protein is present) with lower capacity and higher resolution ones (when less

protein is present) at later stages of purification scheme. The widely used purification methods involve the following steps:

1. Protein extraction, involving

– Extract preparation

– Sub-cellular fractionation

– Solubilization of bound proteins

2. Bulk techniques of protein separation

– Salting out

– Precipitation with organic solvents

– Precipitation with decreased pH or heat

3. Chromatographic techniques











Ion exchange chromatography

Adsorption chromatography

Gel filtration

Affinity chromatography



R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology,

DOI 10.1007/978-1-4419-9785-2_8, # Springer Science+Business Media, LLC 2011



149



150



8



Protein Purification Techniques



4. Electrophoretic procedures

– Native (PAGE) or SDS-PAGE

– Isoelectric focusing

– Two-dimensional gel electrophoresis (2D-electrophoresis)



8.1



Protein Extraction Procedure



The first steps of a typical protein isolation procedure usually consist of washing

the tissue and applying the lysis method. The method should be efficient to disrupt

the cells to release the protein in soluble form of intracellular compartments into a

solution of well-defined composition. The selection of an appropriate buffer is

important in order to maintain the protein at the desired pH and to ensure reproducible experimental results. A 50–100 mM buffer is generally suitable for extraction.

Along with the buffer, other components like chelators, reducing agents, detergents

can be added depending on the nature and location of the protein. Grinding of tissue

with abrasive materials is an effective means of lysis which is achieved by the

abrasive action of grinding the thick paste of sample by hand with alumina or sand.

Then, centrifugation separates the soluble proteins from the membrane fraction and

insoluble debris. Finally, the protein sample may be analysed, further purified or

stored for further use.

1. Materials, equipment and solutions

(a) Prechilled pestle and mortar

(b) Quartz sand or alumina

(c) Extraction buffer: 0.1 M sodium acetate buffer (pH 5.2) containing 12 mM

b-mercaptoethanol

(d) Polyvinyl polypyrrolidone

(e) Muslin cloth

(f) Refrigerated centrifuge

(g) Centrifuge tubes

(h) Beaker and funnel

2. Protocol

(a) Weigh about 40 g of fresh leaf tissue or 25 g of dry leaf tissue and homogenize with 150 mL of extraction buffer (1:4 or 1:6 w/v) by addition in

increments in a chilled pestle and mortar. Add 12 mg of polyvinyl

polypyrrolidone during grinding to remove the phenolics. A pinch of quartz

sand can be added to facilitate thorough grinding.

(b) Squeeze the homogenate through four layers of muslin cloth and clarify the

extract by centrifugation at 12,000 Â g for 10 min.



Tài liệu bạn tìm kiếm đã sẵn sàng tải về

23 Preparations of S-30 Extract for Protein Synthesis In Vitro (Roberts and Paterson 1973)

Tải bản đầy đủ ngay(0 tr)

×