Establishing expression methods for successful crystal of MID962-1200
(2013) MOBY15 20122Degree Projects in Molecular Biology
- Abstract
- Abstract
Moraxella catarrhalis is a new pathogenic, gram negative bacterium that is the cause of many respiratory tract infections. MID was found to be a unique protein with Ig binding capacities. Moraxella catarrhalis immunoglobulin D (IgD)-binding protein (MID) is an outer membrane protein that has specific binding affinity to the soluble cell bound human IgD. A short fragment of MID comprised of 238 amino acid (MID962-1200) contains this IgD binding capacity. MID-deficient Moraxella catarrhalis does not bind to human IgD B cells. MID962-1200 stimulates B cell in the presence of IL-2 and IL-6. The monomeric form of the fragment of the MID protein is about ~200 kDa. The oligomeric form of the fragment has 20 fold stronger binding to... (More) - Abstract
Moraxella catarrhalis is a new pathogenic, gram negative bacterium that is the cause of many respiratory tract infections. MID was found to be a unique protein with Ig binding capacities. Moraxella catarrhalis immunoglobulin D (IgD)-binding protein (MID) is an outer membrane protein that has specific binding affinity to the soluble cell bound human IgD. A short fragment of MID comprised of 238 amino acid (MID962-1200) contains this IgD binding capacity. MID-deficient Moraxella catarrhalis does not bind to human IgD B cells. MID962-1200 stimulates B cell in the presence of IL-2 and IL-6. The monomeric form of the fragment of the MID protein is about ~200 kDa. The oligomeric form of the fragment has 20 fold stronger binding to IgD than the monomer from. The truncated MID962-1200 is efficiently binding to the IgD serum and purified IgD(κ) and IgD(λ) but not to the IgM,IgG,IgA myeloma sera. In this paper we examined the expression and purification of MID962-1200 by the affinity purification and gel filtration. Subsequent crystallization is done by the Hanging drop methods.
Popular science summary:
Despite the fact that the M.catarrhalis bacterium is often considered as a commensal rather harmless bacterium, it is reclassified as a pathogenic gram negative bacterium that mainly causes otitis media and sinusitis in children as well as chronic obstructive pulmonary disease (COPD) and lower respiratory tract infections in adult. One of the outer membrane protein of M.catarrahalis is called M.catarrahalis immunoglobulin D binding protein (MID) that has a strong binding affinity to the soluble cell bound human IgD. The total sequence of MID is 2123 amino acid residues. A short fragment of MID, comprised of 238 amino acid (MID962-1200), contains this IgD binding capacity. MID962-1200 stimulates B cells and secretes IL-2 and IL-6.
MID962-1200 is expressed in E.coli BL21 cells and the clone contains a GST tag and a His tag to ease purification and increase the solubility. The GST tag and His tag allow purification on glutathione sepharose and Ni-NTA respectively. The advantage of using a GST-tagged protein is that the GST affinity tag permits a mild purification process that does not affect the protein native structure and function and preserves antigenicity. However all the tags whether large or small have the potential to interfere with biological activity of protein and may impede crystallization. For the crystallization of MID962-1200 we used a highly specific and efficient protease, the tobacco etch virus (TEV) to cleave of the tags. Thus the recombinant fusion protein is cleaved at a TEV cleavage site between the MID962-1200 fragment and the GST-tag. So finally the GST tag is separated from the protein by exploiting its His-tag in Ni-NTA chromatography.
The crystallization of the protein was performed mainly by hanging drop vapor diffusion. In vapor diffusion a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuse out of the drop until the osmolarity of drop and the precipitant are equal. Hits were found initially in commercially available screen (PACT premier) with conditions 20 % w/v Polyethylene glycol 3350 and 200 mM potassium thiocyanate. For the stabilizing the protein we used Diaminooctane from an additive screen. Then we tried to optimize the reservoir condition by varying potassium thiocyanate 100 mM to 500 mM and Polyethylene glycol 3350 from 15% w/v to 30%w/v. Good crystals were grown at the condition of 23% w/v polyethylene glycol 3350 and 250 mM potassium thiocyanate.
In this experiment we expressed and purified two different types of protein. We were able to purify the protein to the required level of purity for the crystallization.
Advisor: Marjolein Thunnissen
Degree project: 30 credits in Cell and molecular Biology, 2013.
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/3614515
- author
- Hossain, Mohammad Arman
- supervisor
- organization
- course
- MOBY15 20122
- year
- 2013
- type
- H1 - Master's Degree (One Year)
- subject
- language
- English
- id
- 3614515
- date added to LUP
- 2013-03-20 14:01:57
- date last changed
- 2013-03-26 13:40:09
@misc{3614515,
abstract = {{Abstract
Moraxella catarrhalis is a new pathogenic, gram negative bacterium that is the cause of many respiratory tract infections. MID was found to be a unique protein with Ig binding capacities. Moraxella catarrhalis immunoglobulin D (IgD)-binding protein (MID) is an outer membrane protein that has specific binding affinity to the soluble cell bound human IgD. A short fragment of MID comprised of 238 amino acid (MID962-1200) contains this IgD binding capacity. MID-deficient Moraxella catarrhalis does not bind to human IgD B cells. MID962-1200 stimulates B cell in the presence of IL-2 and IL-6. The monomeric form of the fragment of the MID protein is about ~200 kDa. The oligomeric form of the fragment has 20 fold stronger binding to IgD than the monomer from. The truncated MID962-1200 is efficiently binding to the IgD serum and purified IgD(κ) and IgD(λ) but not to the IgM,IgG,IgA myeloma sera. In this paper we examined the expression and purification of MID962-1200 by the affinity purification and gel filtration. Subsequent crystallization is done by the Hanging drop methods.
Popular science summary:
Despite the fact that the M.catarrhalis bacterium is often considered as a commensal rather harmless bacterium, it is reclassified as a pathogenic gram negative bacterium that mainly causes otitis media and sinusitis in children as well as chronic obstructive pulmonary disease (COPD) and lower respiratory tract infections in adult. One of the outer membrane protein of M.catarrahalis is called M.catarrahalis immunoglobulin D binding protein (MID) that has a strong binding affinity to the soluble cell bound human IgD. The total sequence of MID is 2123 amino acid residues. A short fragment of MID, comprised of 238 amino acid (MID962-1200), contains this IgD binding capacity. MID962-1200 stimulates B cells and secretes IL-2 and IL-6.
MID962-1200 is expressed in E.coli BL21 cells and the clone contains a GST tag and a His tag to ease purification and increase the solubility. The GST tag and His tag allow purification on glutathione sepharose and Ni-NTA respectively. The advantage of using a GST-tagged protein is that the GST affinity tag permits a mild purification process that does not affect the protein native structure and function and preserves antigenicity. However all the tags whether large or small have the potential to interfere with biological activity of protein and may impede crystallization. For the crystallization of MID962-1200 we used a highly specific and efficient protease, the tobacco etch virus (TEV) to cleave of the tags. Thus the recombinant fusion protein is cleaved at a TEV cleavage site between the MID962-1200 fragment and the GST-tag. So finally the GST tag is separated from the protein by exploiting its His-tag in Ni-NTA chromatography.
The crystallization of the protein was performed mainly by hanging drop vapor diffusion. In vapor diffusion a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuse out of the drop until the osmolarity of drop and the precipitant are equal. Hits were found initially in commercially available screen (PACT premier) with conditions 20 % w/v Polyethylene glycol 3350 and 200 mM potassium thiocyanate. For the stabilizing the protein we used Diaminooctane from an additive screen. Then we tried to optimize the reservoir condition by varying potassium thiocyanate 100 mM to 500 mM and Polyethylene glycol 3350 from 15% w/v to 30%w/v. Good crystals were grown at the condition of 23% w/v polyethylene glycol 3350 and 250 mM potassium thiocyanate.
In this experiment we expressed and purified two different types of protein. We were able to purify the protein to the required level of purity for the crystallization.
Advisor: Marjolein Thunnissen
Degree project: 30 credits in Cell and molecular Biology, 2013.
Department of Biology, Lund University}},
author = {{Hossain, Mohammad Arman}},
language = {{eng}},
note = {{Student Paper}},
title = {{Establishing expression methods for successful crystal of MID962-1200}},
year = {{2013}},
}