Conjugation and release of Escherichia coli surface displayed Affibody molecules by eSrtA

Abstract

Antibodies have for long time dominated the field of affinity proteins in example imaging and therapeutics, due to their high specificity and affinity. However, they have some major drawbacks such as high molecular weight, complex structure leading to instability and high production cost. Therefore, other types of affinity proteins have been engineered, for example the Affibody molecule which is a small stable three helical protein. One method used to isolate Affibody molecule binders to targets, is to create a large Escherichia coli (E. coli) library with surface displayed Affibody molecules and select binders using FACS (Fluorescence Activated Cell Sorting). To characterize isolated binders from FACS today takes time, subcloning of the proteins of interest is needed before downstream analysis is possible. In this master thesis project it was examined whether transpeptidase eSrtA, a transpeptidase opening up a new world of conjugations difficult to create with other methods, could conjugate three different N- terminal triglycine molecules (functional groups) to E. coli surface displayed Affibody molecules with eSrtA recognition site, LPETG, and release the conjugate from the cell surface. The conjugated N-terminal triglycine molecule is a functional group, enabling direct downstream analysis of the Affibody molecule. eSrtA was recombinant expressed for reaction with two substrates, N-terminal triglycine molecules and surface displayed LPETG-tagged Affibody molecules. It was concluded that transpeptidation by eSrtA was successful and resulted in a surface released Affibody molecule conjugated to a functional group. The surface modification of the released conjugated Affibody molecule did not affect its binding capacities and it proved to work in common characterization methods. Since subcloning and then labelling could be avoided, the method developed in this master thesis would facilitate and accelerate the process from having isolated binders from a library to perform protein characterization of the released conjugates using the functional group.

Popular Abstract

A new fast and simplified method to find future drugs

Has anyone around you ever suffered from cancer or Alzheimer’s? If so, you know how tough the treatment is, or in worse case there is no treatment. In the future you could get a more effective drug that causes less adverse effects, or a marker that points out where the disease is. But what is this future drug and more importantly, how do you find it?

The future drug is a small protein. Proteins are building blocks of the human body and are created by putting amino acids, there are 20 essential, together. In this project a new method was developed in order to easily find small proteins that could be used as drugs or markers for diseases. The small protein binds specifically to a certain target, just like antibodies. Antibodies are large proteins that defend your body against foreign substances. They do not bind any other molecule except the one they are programmed to bind. But how can they be used as drugs?

When suffering from a disease, there is something wrong in your body. When having cancer, some of your own cells grow more than usual and you get a tumor. The tumor cells are different from normal cells, they have a special reporter that instructs the cells to grow or not, just like parents telling you what to do. When suffering from Alzheimer’s, molecules in your brain start to stick together. This is something they should not do and it disrupts functions in the brain. Let’s call the special reporters or the sticky molecules disease-markers.

Medicines used today affect the entire body, which results in side effects. If you instead could have a drug that binds only the disease-marker, the rest of your body would not be affected. By binding the disease-marker, you could force the cancer cell to stop growing or stop the molecules in the brain from sticking together. Another method is to have something binding only the disease-marker, linked with a molecule that shines very bright. It could bind to the disease-marker and with the shining molecule inform the doctor where the cancer cells or the molecules sticking together are. That marker of diseases could lead to a better treatment. Now the antibodies come into the picture, since they bind the programmed molecule very specifically. However, they are not optimal to use as drugs, and therefore special variants of them have been created. One is the small protein. But how do you find a small protein that only binds to the disease-marker?

In this project, a new method to faster and easier find small proteins binding disease-markers was developed from an existing method. The existing method starts with gene manipulation of bacteria, telling the bacteria to produce the small protein and store it on their surface. The bacteria therefore serve as a producer and carrier of the small protein. Secondly, you generate millions of bacteria that all have a unique small protein on their surface. All bacteria with small proteins bound on their surface are then mixed with the disease-marker and a machine picks out the small proteins that can bind to it. The tricky thing now is that the small protein must be separated from the bacteria. You need to separate them to know more about the small protein before it can be used as a drug. The method used now to separate them, takes a lot of time and work, something that we wanted to avoid.

We have developed a method to superfast separate the small protein from the bacteria. We have used something that could resemble a scissor and glue kit. It directly cuts away the small protein from the bacteria after they are picked out by the machine. At the same time, a new molecule is glued on. The glued on molecule simplifies the analysis of the small protein. Thereby, we have created a faster and more simple way for researchers to find new small proteins and at the same time improve their analysis process. Researchers could therefore use this short cut to save time and energy when trying to find new drugs or markers for diseases.

• author: Sjöstrand, Nanna
• supervisor: Ken G Andersson; Johan Svensson Bonde
• organization: Pure and Applied Biochemistry
• course: KBK820 20171
• year: 2017
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry; Technology and Engineering; Biology and Life Sciences
• keywords: Sortase, surface display, Affibody molecules, E. coli library, conjugation and surface release, applied biochemistry, tillämpad biokemi
• language: English
• id: 8913814
• date added to LUP: 2017-09-19 14:46:11
• date last changed: 2017-09-19 14:46:11