A method for in vitro synthesis of unglycosylated recombinant complement component C9

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• The human complement system is crucial for innate immunity.
• Complement component C9 (C9) is essential for the formation of the membrane attack complex.
• Understanding C9 function requires the availability of purified normal and mutant proteins.

Purpose of the Study:

• To establish an in vitro method for synthesizing unglycosylated human C9 proteins.
• To enable the generation of both normal and mutant C9 variants.
• To facilitate rapid screening of mutations impacting C9 biological activity and polymerization.

Main Methods:

• Utilized one or two-step polymerase chain reaction (PCR) to introduce T7 RNA polymerase promoter and mutations into C9 cDNA.
• Performed in vitro transcription of cDNA using T7 RNA polymerase to produce mRNA.
• Translated mRNA in rabbit reticulocyte lysate or wheat germ systems to synthesize C9 protein.
• Confirmed synthesis via agarose gel electrophoresis, mRNA labeling with [alpha-32P]UTP, and protein labeling with [35S]methionine.

Main Results:

• Successfully synthesized unglycosylated recombinant C9 protein with correct molecular mass.
• Wheat germ extract yielded up to 1.5 micrograms of recombinant C9.
• The synthesized unglycosylated C9 exhibited at least 10% of native C9's hemolytic activity.
• Uglycosylated C9 showed increased polymerization compared to native C9.
• Deletion of the first 23 amino acids or mutation of cysteines at positions 33 and 36 enhanced C9 polymerization.

Conclusions:

• Developed a rapid and efficient in vitro synthesis method for unglycosylated C9.
• The method allows for the generation and analysis of C9 mutants.
• This approach is valuable for studying the structure-function relationships of pore-forming proteins like C9.