Technical Field

The present invention relates to a method for producing filtering biofilms produced with a modified bacterial major biofilm protein that can bind with a modified lectin protein exhibiting a high binding affinity to multiple mannose groups in virus envelope glycoproteins of target viruses.

Background of the Invention

Today, the rapid spread of SARS-Cov-2 and many similar viruses has led to a health crisis on a global scale and effects of the said health crisis slows down many sectors. The most accurate approach for preventing the high mutation rates of viral diseases and the permanent damages that are caused by viral diseases encountered for the first time in the human body is observed as prevention of the said viruses from entering the human body; therefore, air filters effective against biological factors are used in ventilation systems in crowded environments and in areas with high virus circulation.

However, due to the fact that the said air filters are not produced for the purpose of filtering certain types of viruses actively and ensuring that all components below a certain pore size are released back into the air circulation in an uncontrolled way, it is not possible to effectively prevent SARS-Cov-2 viruses and many similar viruses that can spread rapidly in a way to cause an epidemic. Considering the deficiencies of air filters that are effective against biological factors in the current technique, there is a need in the state of the art for a method for producing filtering biofilms produced with a modified bacterial major biofilm protein that can bind with a modified lectin protein exhibiting a high binding affinity to multiple mannose groups in virus envelope glycoproteins of target viruses.

The International patent document no. W02014008406A2, an application in the state of the art, discloses a compound which enables to use bacterial biofilm matrix for protein targeting and a method for producing the said compound. The bacterial biofilm is regulated by means of a heterologous molecule that is associated with the said target bacterial biofilm or binds to the bacterial biofilm, and the use of biofilm fibers and/or bacteria with modified biofilms are ensured for drug and vaccine targeting, end product purification in biotechnological processes and chemical dissolution in bioprocessing processes.

Summary of the Invention

The objective of the present invention is to realize a method for producing filtering biofilms produced with a modified bacterial major biofilm protein that can bind with a modified lectin protein exhibiting a high binding affinity to multiple mannose groups in virus envelope glycoproteins of target viruses.

Detailed Description of the Invention

“Method for Producing Virus-Filtering Modified Lectin-Binding Modified Biofilm” realized to fulfil the objective of the present invention is shown in the figures attached, in which:

Figure l is a flowchart of the inventive device. The components illustrated in the figures are individually numbered, where the numbers refer to the following:

100. Method

The inventive method (100) for producing filtering biofilms produced with a modified bacterial major biofilm protein that can bind with a modified lectin protein exhibiting a high binding affinity to multiple mannose groups in virus envelope glycoproteins of target viruses comprises the following steps:

- sequencing the marker peptide and the marker peptide-recognizing protein by modifying the targetable quaternary protein which forms an isopeptide bond (101);

- creating a modified bacterial major biofilm protein sequence by inserting the determined marker peptide sequence into the bacterial major biofilm protein sequence (102);

- creating a modified lectin protein sequence by inserting the determined marker peptide-recognizing protein sequence into the sequence of the lectin protein which recognizes the target virus with a high binding affinity (103);

- proliferating the created modified bacterial major biofilm protein sequence and the modified lectin protein sequence in appropriate microorganisms which are grown in appropriate media (104);

- purifying the proliferated modified bacterial major biofilm protein by means of filtration technique (105);

- purifying the proliferated modified lectin protein by means of immobilized metal ion affinity chromatography (106);

- producing a modified lectin-bound modified bacterial major biofilm protein complex by linking the purified modified bacterial major biofilm protein with the purified modified lectin protein under appropriate conditions by means of an isopeptide bond between the marker peptide and the marker peptide-recognizing protein (107);

- creating a virus-filtering biofilm filter by forming fibers from a modified lectin-bound modified bacterial major biofilm protein complex under appropriate conditions (108).

The step of sequencing the marker peptide and the marker peptide-recognizing protein by modifying the targetable quaternary protein which forms an isopeptide bond (101) included in the inventive the method (100) is configured to modify at least one protein with a targetable quaternary structure, that can form an isopeptide bond between thereof to create a compatible molecular pair that can attach to a specific surface by inserting one part into a molecule with filtering ability and the other part thereof into biofilm polymers, such that it can bind to other molecules and to remove the sequences of the modified protein fragments. In one preferred embodiment of the invention, the step of sequencing the marker peptide and the marker peptide-recognizing protein by modifying the targetable quaternary protein which forms an isopeptide bond (101) is configured to obtain the SpyTag marker peptide and the SpyCatcher marker peptide-recognizing protein by cleaving a region included in the FbaB protein of Streptococcus pyogenes bacteria into two parts and modifying them, and to sequence the obtained SpyTag and SpyCatcher molecules.

The step of creating a modified bacterial major biofilm protein sequence by inserting the determined marker peptide sequence into the bacterial major biofilm protein sequence (102) included in the inventive the method (100) is configured to insert the isopeptide-bonding marker peptide sequence to the bacterial major film protein sequence selected to create a capturable position on the biofilm. In one preferred embodiment of the invention, the step of creating a modified bacterial major biofilm protein sequence by inserting the determined marker peptide sequence into the bacterial major biofilm protein sequence (102) is configured to create a modified bacterial major biofilm protein sequence by inserting the SpyTag marker peptide sequence into the sequence of the CsgA major biofilm protein of Escherichia coli bacteria.

The step of creating a modified lectin protein sequence by inserting the determined marker peptide-recognizing protein sequence into the sequence of the lectin protein which recognizes the target virus with a high binding affinity (103) included in the inventive the method (100) is configured to insert the marker peptide-recognizing protein sequence that can form isopeptide bond, into the sequence of at least one lectin protein that recognizes the target virus with a high binding affinity. In one preferred embodiment of the invention, the step of creating a modified lectin protein sequence by inserting the determined marker peptide-recognizing protein sequence into the sequence of the lectin protein which recognizes the target virus with a high binding affinity (103) is configured to create a modified lectin protein sequence by inserting the SpyCatcher marker peptide-recognizing protein sequence into the sequence of the Griffithsin protein isolated from red algae of the genus Griffithsia that has been identified as having a quite high binding affinity to the SARS-CoV-2 virus.

The step of proliferating the created modified bacterial major biofilm protein sequence and the modified lectin protein sequence in appropriate microorganisms which are grown in appropriate media (104) included in the inventive method (100) is configured to insert the modified bacterial major biofilm protein sequence and the modified lectin protein sequence into at least one target bacterium by at least one of the horizontal gene transfer techniques, and to produce modified bacterial major biofilm proteins and modified lectin proteins by cultivating the sequence inserted target bacteria in appropriate media. In one preferred embodiment of the invention, the step of proliferating the created modified bacterial major biofilm protein sequence and the modified lectin protein sequence in appropriate microorganisms which are grown in appropriate media (104) is configured to produce SpyTag-CsgA modified bacterial major biofilm protein and SpyCatcher-Griffithsin modified lectin protein in different bacterial colonies.

The step of purifying the proliferated modified bacterial major biofilm protein by means of filtration technique (105) included in the inventive method (100) is configured to purify the modified bacterial major biofilm protein, that is created by the target bacteria produced in the medium, by separating it from the medium by means of filtration technique. In one preferred embodiment of the invention, the step of purifying the proliferated modified bacterial major biofilm protein by means of filtration technique (105) is configured to purify the SpyTag-CsgA modified bacterial major biofilm protein, that is produced in the medium, by separating it from the medium by means of filtration technique.

The step of purifying the proliferated modified lectin protein by means of immobilized metal ion affinity chromatography (106) included in the inventive method (100) is configured to remove the modified lectin protein, that is created by the target bacteria produced in the medium, from other components in the medium by means of immobilized metal ion affinity chromatography technique. In the preferred embodiment of the invention, the step of purifying the proliferated modified lectin protein by means of immobilized metal ion affinity chromatography (106) is configured to remove the SpyCatcher-Griffithsin modified lectin protein, that is created by the target bacteria produced in the medium, from other components in the medium by means of immobilized metal ion affinity chromatography technique.

The step of producing a modified lectin-bound modified bacterial major biofilm protein complex by linking the purified modified bacterial major biofilm protein with the purified modified lectin protein under appropriate conditions by means of an isopeptide bond between the marker peptide and the marker peptide- recognizing protein (107) included in the inventive method (100) is configured to link the modified lectin protein to the modified bacterial major biofilm protein by spontaneously-formed isopeptide bonds by combining the purified modified bacterial major biofilm protein with the purified modified lectin protein. In one preferred embodiment of the invention, the step of producing a modified lectin- bound modified bacterial major biofilm protein complex by linking the purified modified bacterial major biofilm protein with the purified modified lectin protein under appropriate conditions by means of an isopeptide bond between the marker peptide and the marker peptide-recognizing protein (107) is configured to produce the modified lectin-bound modified bacterial major biofilm protein complex designed to capture the SARS-CoV-2 virus by inserting the purified SpyCatcher- Griffithsin modified lectin protein into the purified SpyTag-CsgA modified bacterial major biofilm protein.

The step of creating a virus-filtering biofilm filter by forming fibers from a modified lectin-bound modified bacterial major biofilm protein complex under appropriate conditions (108) included in the inventive the method (100) is configured to create fibers under suitable polymerization conditions for a modified lectin-bound modified bacterial major biofilm protein complex and then to turn these fibers into virus-filtering biofilm filters containing these fibers. In one preferred embodiment of the invention, the step of creating a virus-filtering biofilm filter by forming fibers from a modified lectin-bound modified bacterial major biofilm protein complex under appropriate conditions (108) is configured to create fibers under appropriate polymerization conditions for the modified lectin- bound modified bacterial major biofilm protein complex created with SpyCatcher- Griffithsin combined with SpyTag-CsgA and then to turn these fibers into virusfiltering biofilm filters containing these fibers.

Industrial Application of the Invention In the inventive method (100), the SpyTag marker peptide and the Spy Catcher marker peptide-recognized protein are sequenced by creating the SpyTag marker peptide and the SpyCatcher marker peptide-recognized protein by modifying the FbaB protein of Streptococcus pyogenes which is a targetable quaternary structured protein that forms the isopeptide bonds (101); the SpyTag-CsgA modified bacterial major biofilm protein sequence is created by inserting the determined SpyTag marker peptide sequence into the sequence of the CsgA major biofilm protein of Escherichia coli bacteria (102); the SpyCatcher-Griffithsin modified lectin protein sequence is created by inserting the determined SpyCatcher marker peptide-recognizing protein sequence into the Griffithsin protein sequence isolated from red algae of the genus Griffithsia, which was determined to have a quite high binding affinity to SARS-CoV-2 virus (103); the SpyTag-CsgA modified bacterial major biofilm protein sequence and the SpyCatcher-Griffithsin modified lectin protein sequence are proliferated in the target bacteria, which are grown in appropriate media by being inserted into the target bacteria by at least one of the horizontal gene transfer techniques (104); the proliferated SpyTag-CsgA modified bacterial major biofilm protein is purified by means of filtration technique (105); and the proliferated SpyCatcher-Griffithsin modified lectin protein is purified by means of immobilized metal ion affinity chromatography (106); the CsgA-SpyTag-SpyCatcher-Griffithsin modified lectin- bound modified bacterial major biofilm protein complex is produced by spontaneously-formed isopeptide bonding between the marker peptide and the marker peptide-recognizing protein under appropriate conditions, by inserting the purified SpyCatcher-Griffithsin modified lectin protein into the purified SpyTag- CsgA modified bacterial major biofilm protein (107); the virus filtering biofilm filters containing the created fibers are produced by creating fibers from the CsgA-SpyTag-SpyCatcher-Griffithsin modified lectin-bound modified bacterial major biofilm protein complex under appropriate conditions (108). The inventive method (100) enables to produce filters that separate viruses such as SARS-CoV-2, which have high propagation rates and persistent effects, from air masses by molecular binding affinity-based filtering in order to reduce the infection rate.

Within these basic concepts; it is possible to develop various embodiments of the inventive “Method for Producing Virus-Filtering Modified Lectin-Binding Modified Biofilm (100)”; the invention cannot be limited to examples disclosed herein and it is essentially according to claims.