AFFINITY PURIFICATION AND REFOLDING ANALYSIS

OF

By Seat No : 2061

hPI

Under the guidance of Dr. Sanjay Sonar, Biogenomics Ltd.

INTRODUCTION

Introduction to insulin.

Introduction to protein purification.

Background to Insulin purification strategies.

STEP WISE METHOD USEDAffinity purification by IMAC.

Protein Quantification by Uv-Vis Spectroscopy.

Visualization of the protein on SDS- PAGE.

Confirmation of the protein on MALDI-TOF-MS.

Dialysis

Refolding

Dialysis

Refolding analysis by HPLC.

AIM AIM To produce commendable amounts of the purified

and active recombinant human proinsulin molecule at an optimum rate in a cost- effective manner.

OBJECTIVES Solubilisation of the wet cell mass of the 7K

recombinant human pro insulin inclusion bodies. To purify the 6-Histidine tagged protein by

immobilized metal ion affinity chromatography using Ni+2 -NTA resin.

Visualization of the protein on SDS –PAGE and MALDI-TOF-MS.

Refolding analysis by HPLC.

METHODS Preparation of the sample for affinity

purification.

WCM containing expressed protein was dissolved in 0.02M Tris pH 8.0

 It was digested with lysozyme.  The disrupted cells were then centrifuged.   Protein/ Inclusion body pellet was thus obtained.  The pellet was washed with 8M urea and 0.02M

Tris pH 8.0. Centrifugation of the pellet at 8000 rpm for 20

min at 4⁰C The pellet obtained is solubilised prior to its

loading on IMAC 

METHODS AFFINITY PURIFICATION OF THE SAMPLEPacking of the column.

By pass of the column.

CIP of the column.

Charging of the column using 0.2N NiSO4.

Loading of crude FP rhPI sample onto column. Buffer used- 8M Urea, 0.02M Tris-HCl, pH 8.5

Elution.Buffer used- 8M urea, 0.02M TRIS-HCl and 0.5M

imidazole

METHODS Protein quantification By UV-Vis

spectroscopy The wavelength spectrum was adjusted in uv

range from 200-400nm. For the loading sample, flow through, 7% B and

30% B, binding buffer was adjusted as blank. For the 100% B fraction, elution buffer was used

as blank. The concentration of the protein and the total

protein was calculated by the following formula: Concentration :- (O.D at 280nm- O.D at

320nm)/extinction coefficient X dilution factor Total protein :- Concentration X Total volume.

METHODS

SDS PAGE Assembly of the electrophoresis unit.

15% resolving gel and 5% stacking gel was used.

Preparation of the sample for loading.

The Run

The gel was run at 7 volts for 4 hours in the tank buffer at a pH of

Staining of the sample

The sample was stained by using silver staining method.

METHODS

MALDI-TOF-MS

Loading of sample on the target plate

1 µl of the sample was mixed with equal quantity of sinapinic acid and loaded onto the target plate.

Crystallisation of the protein.

Loading the plate onto the instrument and

obtaining spectra.

METHODSREFOLDING ANALYSIS BY HPLC

Preparation of the sample using TFA.

Preparation of the HPLC unit.

Loading the sample.

Elution.

RESULTS AP RUN 1

30%

RESULT AP RUN 2

30%

RESULTQuantification by u-v spectrophotometer

Ni+2-NTA Run hPI sample. (Run 1)

RESULTNi+2-NTA Run hPI sample (Run 2)

RESULT Visualisation of the protein on SDS-PAGE

1. MWM Medium2. Loading sample3. Flow through

(2µl)4. Flow through

(4µl)5. 7% (1)6. 7% (2)7. 30% (1)8. 30% (2)9. 100%

26 22

18

14

RESULT MALDI-TOF-MS

14 KDa

RESULT REFOLDING ANALYSIS BY HPLC hPI Refolding O hr

RESULT hPI Refolding 5 hr

DISCUSSION

Various factors related to Affinity Purification Proper optimization of the parameters like column

length, concentration of the sample, the buffers used, flow rate with respect to the amount of sample that has been loaded has o be carried out.

SDS-PAGE

In case of the results of SDS-PAGE, the gel has been run to check that no protein is being eluted out in the flow through establishing that the binding buffers used for AP are well optimized.

Dialysis

Dialysis of the sample has to be carried out after and before refolding reaction is set. Based on the amount of sample, dialysis, ultrafiltration or GPC can be carried out.

CONCLUSION

Solubilisation of the wet cell mass of the 7K recombinant human pro insulin inclusion bodies was carried out using 8M Urea.

The 6-His tagged protein was purified by immobilized metal ion affinity chromatography using Ni+2 -NTA resin.

The presence of the purified protein in the eluate was confirmed by SDS –PAGE and MALDI-TOF-MS.

Refolding analysis by HPLC was performed.

REFERENCES

Luis Felipe Vallejo and Ursula Rinas, Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins, 2004 September 2. doi: 10.1186/1475-2859-3-11.

"Recombinant DNA technology in the synthesis of human insulin". Little Tree Publishing. http://www.littletree.com.au/dna.htm.Retrieved 2009-11-03.

ACKNOWLEDGEMENTS

My project work would have been indeed incomplete with out the guidance of

Dr Sanjay Sonar, Dr Archana Krishnan,

Mr Nikhil Ghade and the entire staff of Biogenomics Ltd.

As also Ms. Shilpa Bhingarde, Ms. Priyanka Bapat- my guides at G.N.Khalsa college.