Purification Of Recombinant Protein Biology Essay

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Affinity chromatography separates proteins by their binding specificities. The proteins retained on the coloumn are those that bind specifically to a ligand cross linked to the beads. Affinity chromatography is used in protein purification, and protein engineering methods, it will work based on the biological interactions and commonly used in fractionating the macromolecules. This method is not only for the protein purification but also for polysaccharides, glycoproteins, nucleic acids etc. In recombinant DNA technology different types of fusion proteins are produced from prokaryotes and eukaryotes. Affinity chromatography is used for parallel purification of protein at a time. Gene should be designed according to our suitable protein. The gene should have 6 histidine coded codons. The histidine has affinity with Nickel column; based on the affinity we can separate our desired protein from the mixture. (Deyl. Z et al.1979)

SDS-PAGE is the most widely used method for qualitatively analysing protein mixtures. It is particularly useful in monitoring of protein purification. This method is based on the separation of proteins according to their size, the method can also be used to determine the molecular mass of proteins. In this experiment we are going to purify LH3 protein by using Ni-NTA matrix. The SDS-PAGE is used to analyse the purified protein and determine the molecular weight. The PAGE is used to separate the proteins by their denaturation character. If we place the denatured protein in to an electric field, all the proteins move towards the positive pole at the same rate, no separation by size. Because of this reason we are going to put the proteins in to different environment which is going to separate the proteins based on their size. The environment of choice is polyacrylamide, which is polymer of acrylamide monomers. (Laemmli, U.K. 1970)

Fig 1: SDS-PAGE [1] Fig 2: Affinity chromatography [2]

Links:

http://www.google.com/images?um=1&hl=en&biw=1294&bih=581&tbs=isch%3A1&sa=1&q=sds+page&aq=f&aqi=g7&aql=&oq=&gs_rfai=

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PROCEDURE:

The experiments were performed according to the protocol described in Biochemistry methods I, practical course (Salin M., 2007). Staining was done by using comassie brilliant blue dyeing in the microwave 30 min. The excess coloured was removed by decolouring solution for 48 hrs. The photograph was taken for measuring drifts; it was shown in fig-3.

RESULT:

Recombinant protein was purified by using Ni-NTA affinity chromatography and analysed by SDS-PAGE. Recombinant protein induction was done by using the IPTG. SDS-PAGE was run against positive pole for 1 hr at 200 volts, after this the gels were stained by comassie brilliant blue, the bands were visualized after destaining the gels. Standard protein distance was calculated based on their molecular weights; the standard curve was drawn that shows in Graph 1&2. Molecular weights & drifts of standards, samples (6-13) were shown in table-1.

Table 1:The molecular weights of the standards and strongest bands of samples 6-13.

Sample

Molecular weight

(Da)

Log Mp

Drift (mm)

gel 1

Drift (mm)

gel 2

Standard

118000

5.07

6

8

90000

4.95

12

16

50000

4.69

23

30

34000

4.53

33

41

26000

4.41

52

60

19000

4.27

63

68

Samples

6

19420

4.28

58

7

20000

4.30

59

8

20900

4.32

57

9

26000

4.41

54

10

21640

4.33

60

11

21640

4.33

60

12

21640

4.33

60

13

22450

4.35

58

Fig 3: SDS-PAGE gel electrophoresis

Separating gel concentration: 15%

Time: 1 hr â€" 1:30 hr

Running buffer: 1X running buffer, Composition is 25 mM Tris, 0.192 M glycine and 0.1 SDS.

Visualizing agent: comassie Brilliant blue

Graph 1: x-axis log Mp & y- axis drift.

Graph 2: x-axis log Mp & y- axis drift.

CONCLUSION:

The aim of this experiment is to purify a recombinant protein produced by bacteria by using affinity chromatography. IPTG is used to induce the recombinant protein production in bacterial cells. The characteristics of protein were studied by SDS-PAGE. IPTG takes 3 â€" 5 hr at 38°C to induce the recombinant protein production. Lysate matrix has thousands of proteins when we incubate this at cold room for 1 hour recombinant proteins (with His-tag) were bound to the provided matrix.

Based on pH variation we can elute the desired proteins from mixture. All bacterial proteins (NOT recombinant proteins) were eluted at high pH buffer. Only recombinant proteins were eluted at lower pH, it means the pH 4.5 is the suitable pH for elutes the recombinant proteins with His-tag. In sample 1 there is no recombinant protein because it was collected before the addition of IPTG. Sample 2 has recombinant protein it was confirmed by the bright band in figure-3. The size of the recombinant protein was 22Kda. Final samples (9, 10, 11, 12, and 13) have recombinant protein because of their brightest bands. (Shown in Fig-3)

For the case book:

Ampicillin is a semi-synthetic derivative of penicillin that weakens the peptideglycan cross linking and thus inhibits cell wall synthesis.

Sterile working means avoiding the contamination. All equipments should be used in micro-organism free.

Nickel â€"NTA has affinity with histidine, which is present in the protein. In affinity chromatography the histidine amino a acid is used as affinity agent.

Proteins are very sensitive to their solvent environment. Urea is a common chemical denaturant of proteins. Urea disrupts non-covalent bonds in the proteins.

APS is an initiator for gel formation. TEMED used in SDS-PAGE for Chemical polymerisation of acrylamide gel.

Glycerol is a weighing agent and also preservative agent. It also helps to weigh down the sample into the wells without being spread while loading.

Reducing agent is used to disrupt disulfide bonds to ensure the protein is fully denatured before loading on the gel, ensuring the protein runs uniformly.

Lysate matrix has thousands of proteins when we incubate this at cold room for 1 hour recombinant proteins was bound to the provided matrix.

Sample 5 contains the proteins which not tightly bind to the matrix pack.

Solutions can be sterilized by heat. This can be two types one is dry heat sterilization and second is moisture sterilization. Example for dry heat sterilization is flaming and example for moisture heat sterilization is autoclave. Chemical methods and radiation methods are also available.

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