Search

CN-122011151-A - High-efficiency expressed soluble serum amyloid A mutant and preparation method and application thereof

CN122011151ACN 122011151 ACN122011151 ACN 122011151ACN-122011151-A

Abstract

The invention relates to a Serum Amyloid A (SAA) protein mutant and a preparation method thereof. Specifically, the serum amyloid A protein mutant contains core amino acid selected from the group consisting of (a) amino acid at 1 st position is A or G, (b) amino acid at 5 th position is A or G, and/or (c) amino acid at 8 th position is A, wherein the amino acid position numbers are based on any sequence shown in SEQ ID NO 1 and 69-73. The method of the present invention increases SAA protein production and avoids endotoxin problems and differences in folding or immunogenicity when using other systems to express proteins.

Inventors

  • YE DEQUAN
  • CHANG YIXIN

Assignees

  • 香港中文大学（深圳）
  • 香港中文大学(深圳)福田生物医药创新研发中心

Dates

Publication Date
20260512
Application Date
20250917

Claims (10)

  1. 1. A serum amyloid a protein mutant, characterized in that the serum amyloid a protein mutant is a non-native protein and comprises a core amino acid selected from the group consisting of: (a) Amino acid at position 1 is A or G; (b) Amino acid at position 5 is A or G, and/or (C) Amino acid at position 8 is A; wherein the amino acid position numbers are based on any one of the sequences shown in SEQ ID NO 1 and 69-73.
  2. 2. The serum amyloid a protein mutant according to claim 1, wherein the serum amyloid a protein mutant has the same or substantially the same sequence as shown in SEQ ID No.1 except for amino acids 1, 5 and 8.
  3. 3. The serum amyloid a protein mutant according to claim 1, wherein the serum amyloid a protein mutant is a non-native protein and comprises a core amino acid selected from the group consisting of: (a1) Amino acid at position 1 is A; (a2) Amino acid at position 5 is A; (a3) Amino acid at position 1 is G; (a4) The 5 th amino acid is G; (a5) Amino acid at position 1 is A and amino acid at position 5 is G; (a6) Amino acid at position 1 is G and amino acid at position 5 is A; wherein the amino acid position numbers are based on any one of the sequences shown in SEQ ID NO 1 and 69-73.
  4. 4. The serum amyloid a protein mutant according to claim 1, wherein the serum amyloid a protein mutant is a non-native protein and comprises a core amino acid selected from the group consisting of: (a1) Amino acid at position 1 is A; (a5) Amino acid at position 1 is A and amino acid at position 5 is G; (a6) Amino acid at position 1 is G and amino acid at position 5 is A; wherein the amino acid position numbers are based on any one of the sequences shown in SEQ ID NO 1 and 69-73.
  5. 5. The recombinant protein is characterized by having a structure shown in a formula I or combining Z1-Z6 in any order: Z1-Z2-Z3-Z4-Z5-Z6(I) Wherein Z1 is absent or a translation initiation regulatory element; Z2 is a signal peptide; z3 is an affinity purification tag; z4 is a solubility enhancing and chaperone tag; Z5 is a specific protease cleavage site; z6 is the serum amyloid a protein mutant of claim 1.
  6. 6. The recombinant protein according to claim 5, wherein said translation initiation regulatory element is Kozak or IRES; The affinity purification tag is selected from the group consisting of His tag, FLAG tag, GST tag, strep II tag, or a combination thereof; the solubility enhancing and chaperone tag is selected from the group consisting of SUMO, MBP, trx, nusA, HA tags, or combinations thereof; The site-specific protease is selected from the group consisting of TEV protease, thrombin, enterokinase, preScission, or a combination thereof.
  7. 7. A polynucleotide encoding the serum amyloid a protein mutant of claim 1 or the recombinant protein of claim 5.
  8. 8. A carrier, characterized in that, the vector contains the polynucleotide of claim 7.
  9. 9. A host cell comprising the vector of claim 8 or the polynucleotide of claim 7 integrated into the genome.
  10. 10. A method for preparing the serum amyloid a protein mutant according to claim 1 or the recombinant protein according to claim 5, comprising the steps of: Culturing the host cell of claim 9 under conditions suitable for expression, inducing expression, and purifying to obtain the serum amyloid a protein mutant of claim 1 or the recombinant protein of claim 5.

Description

High-efficiency expressed soluble serum amyloid A mutant and preparation method and application thereof Technical Field The invention relates to the field of protein engineering, in particular to a soluble serum amyloid A mutant with high expression efficiency, a preparation method and application thereof. Background Inflammatory response is the body's own defense mechanism against external injury or internal abnormalities, involving the core functions of the innate immune system, with the aim of eliminating harmful stimuli and promoting repair of damaged tissues. This process is critical in a variety of biological and medical studies, as it involves a wide range of pathological conditions and methods of treatment. In the event of inflammation, significant concentration changes can occur in the serum for specific proteins, particularly Acute Phase Proteins (APP). Serum amyloid a protein (SAA) is one of these acute phase reactive proteins, whose concentration is significantly elevated in both acute and chronic inflammation, and becomes an important biomarker in inflammatory states. The change in concentration of SAA not only plays an indicative role in clinical diagnosis, but also plays a key role in understanding the pathogenesis of inflammation-related diseases. Particularly in chronic diseases like Alzheimer's disease, prolonged elevation of SAA may lead to the formation of Amyloid A (AA) and deposition in major organs and tissues, which in turn causes a serious series of health problems including, but not limited to, organ failure. Therefore, SAA plays an important role in the development of inflammatory responses and chronic diseases, and its research is of great importance for the deep understanding and treatment of these diseases. With the deep knowledge of inflammation and its related diseases, the research on SAA is increasingly demanded. The traditional serum amyloid protein preparation method is usually extracted from human serum, and the method is difficult to purify, has insufficient yield, has risks of cross infection and the like, and is difficult to meet the demands of scientific research or clinical application. In addition, although it has been reported that human serum amyloid can be expressed in microbial cells such as yeast or bacteria, the proteins expressed by these microbial cells differ from the amyloid in human serum in terms of structure, function and immunogenicity. And most of commercialized SAA is prepared by an escherichia coli expression purification system, and endotoxin pollution exists, so that the research on the effect of SAA in diseases is greatly influenced. Therefore, the stable, reliable and efficient preparation method of SAA with low endotoxin can support the wide application of SAA in experimental research and clinical application. The effective SAA preparation not only can improve the accuracy and reproducibility of experiments, but also is helpful for deepening our understanding of the mechanisms of inflammation and related diseases. In addition, high purity and high activity SAA provides an important experimental basis for developing new therapeutic strategies, such as detection of disease markers and identification of drug targets. Therefore, the development of a mammalian cell expression system that is both efficient and safe is currently a problem to be solved. This will not only facilitate basic medical and biological studies, but may also directly affect the development of new therapies and early diagnosis of disease. Such research progress will provide valuable resources to the medical community, providing new possibilities for improving public health and treating related diseases. Disclosure of Invention The invention aims to provide a serum amyloid A mutant with low endotoxin, good water solubility and stability and a preparation method thereof. It is another object of the present invention to provide a host cell that efficiently and safely expresses serum amyloid A. In a first aspect of the invention, there is provided a serum amyloid a protein mutant which is a non-native protein and which comprises a core amino acid selected from the group consisting of: (a) Amino acid at position 1 is A or G; (b) Amino acid at position 5 is A or G, and/or (C) Amino acid at position 8 is A; wherein the amino acid position numbers are based on any one of the sequences shown in SEQ ID NO 1 and 69-73. In another preferred embodiment, the serum amyloid a protein mutant is a non-native protein and comprises a core amino acid selected from the group consisting of: (a1) Amino acid at position 1 is A; (a2) Amino acid at position 5 is A; (a3) Amino acid at position 1 is G; (a4) The 5 th amino acid is G; (a5) Amino acid at position 1 is A and amino acid at position 5 is G; (a6) Amino acid at position 1 is G and amino acid at position 5 is A; wherein the amino acid position numbers are based on any one of the sequences shown in SEQ ID NO 1 and 69-73. In another preferred embodim