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US-12624377-B2 - Systems and method for the production of Griffithsin and related proteins

US12624377B2US 12624377 B2US12624377 B2US 12624377B2US-12624377-B2

Abstract

Methods and kits are provided for producing Griffithsin. The methods include providing a genetically modified microorganism comprising a gene encoding Griffithsin protein operably linked to an inducible promotor and growing the genetically modified microorganism under conditions that induce the promotor and cause expression of griffithisin. The Griffithsin is purified by releasing Griffithsin from the microorganism by cellular disruption, performing a precipitation step to remove contaminating protein and nucleic acids, and performing an anion exchange chromatography step.

Inventors

  • Michael Lynch
  • John Decker

Assignees

  • DUKE UNIVERSITY

Dates

Publication Date
20260512
Application Date
20210319

Claims (10)

  1. 1 . A method of producing Griffithsin comprising: i) providing a genetically modified E. coli microorganism comprising a gene encoding a Griffithsin protein operably linked to an inducible promotor; ii) growing the genetically modified microorganism under conditions that induce the promotor and cause expression of Griffithsin, the inducible promotor expresses the Griffithsin protein when phosphate is depleted from media in which the genetically modified microorganism is growing; iii) releasing Griffithsin from the microorganism by cellular disruption; iv) performing a precipitation step to remove contaminating protein and nucleic acids; v) performing an anion exchange chromatography step, wherein the precipitation step and anion exchange chromatograph step produce purified Griffithsin, and wherein the Griffithsin is a protein of the red algae Griffithsia.
  2. 2 . The method of claim 1 , wherein Griffithsin gene is SEQ ID NO: 1.
  3. 3 . The method of claim 1 , wherein cellular disruption occurs by sonication or cellular lysis.
  4. 4 . The method of claim 1 , wherein the precipitation step is performed at a temperature greater than 55° C. and less than 73° C.
  5. 5 . The method of claim 1 , wherein the precipitation step is performed at a temperature of 55° C.
  6. 6 . The method of claim 1 , wherein the precipitation step further comprises heating to effect precipitation between 5 and 60 minutes.
  7. 7 . The method of claim 1 , wherein the precipitation step further comprises maintaining a pH greater than 2.5 and less than 4.
  8. 8 . The method of claim 1 , wherein the precipitation step occurs in the presence of at least 0.8M (NH 4 ) 2 SO 4 .
  9. 9 . The method of claim 1 , wherein the precipitation step occurs in the presence of between 0.8M and 1.4 M (NH 4 ) 2 SO 4 .
  10. 10 . The method of claim 1 , wherein the precipitation step further comprises a centrifugation step.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62/992,557 filed Mar. 20, 2020, which is incorporated by reference herein in its entirety. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made with Government support under Federal Grant no. 12043956 awarded by the Office of Naval Research, Federal Grant no. EE0007563 awarded by the Department of Energy (DOE), R61 AI140485-01 awarded by the National Institute of Health (NIH/NIAID/DMID), and Federal Grant no. T32GM008555 awarded by the National institute of General Medical Sciences. The Federal Government has certain rights to this invention. SEQUENCE LISTING The instant application contains a Sequence Listing which has been filed electronically in ASCII format as 47381-47_ST25 created Mar. 19, 2021 that is 3,505 bytes in size and is hereby incorporated by reference in its entirety. BACKGROUND Griffithsin, a lectin, has potential to prevent and treat and prevent numerous viruses including HIV, HCV, HSV, SARS-CoV, and SARS-CoV-2. For SARS-CoV-2 prevention and treatment in the current pandemic, annual demand could reach billions of doses and affordability is paramount. SUMMARY The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter. The present disclosure is based, in part, on lab-scale validation of a bioprocess that supports production volumes>20 tons per year (5 billion doses) at costs below $10,000/kg of Griffithsin ($0.04/dose) in a finished antiviral product. Recombinant expression in engineered E. coli enables Griffithsin titers>2.7 g/L. A single precipitation step removing >99% of host cell proteins and virtually all nucleic acids is followed by a single chromatography step removing residual endotoxin leading to pure, active Griffithsin. These results support the potential of biologics in very large scale, low-cost applications such as preventive antivirals against SARS-CoV-2 and highlight the importance of bioprocess innovations in enabling these applications. Accordingly, one aspect of the present disclosure provides a process for the production and purification of Griffithsin or a related protein comprising, consisting of, or consisting essentially of: i) expressing the Griffithsin or the related protein in E. coli under the control of a low phosphate inducible promoter; ii) releasing the Griffithsin or the related protein expressed in (i) from the cell via cellular disruption or lysis; iii) performing a precipitation step to remove contaminating protein and nucleic acids performed at temperatures greater than 55 degrees Celsius, (NH4)2SO4 concentrations greater than 0.8M and a pH less than 4; and vi) performing at least one anion exchange chromatography purification step. In one aspect, the related protein has greater than 60% identity to Griffithsin or consists of multiple domains each having greater than 60% identity to Griffithsin. In another aspect, the temperature during precipitation is greater than 55 degrees Celsius and less than 73 degrees Celsius. In another aspect, the duration of heating to effect precipitation is between 5 and 60 minutes. In another aspect, the concentration of (NH4)2SO4 during precipitation is greater than 0.8 M and less than 1.4 M. In other aspects, the pH during precipitation is greater than 2.5 and less than 4. In another aspect, the concentration of (NH4)2SO4 during anion exchange chromatography is greater than 30 mM and less than 60 mM. In another aspect, the precipitates are removed by centrifugation. In yet another aspect, spray drying is used for the final formulation. Other methods, features and/or advantages is, or will become, apparent upon examination of the following figures and detailed description. It is intended that all such additional methods, features, and advantages be included within this description and are protected by the accompanying claims. BRIEF DESCRIPTION OF DRAWINGS The novel features of the invention are set forth with particularity in the claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative aspects, in which the principles of the invention are used, and the accompanying drawings of which: FIG. 1 is a table summarizing plasmids and microorganism strains used in this study. FIG. 2 is a graph showing the cost and scale for various GRFT manufacturing processes. FIG. 3A-3E are graphs showing E. coli-based fermentation-based processes for GRFT manufacturing. FIG. 4A-4C is a chart showing E. coli-based fermentation-based processes for GRFT manufacturing in accordance with one aspect of the present disclosure. FIG. 5 is a graph showi