(L-468) Recombinant protein S: A step towards protein S production in a heterologous system

Protein S deficiency is an autosomal dominant disorder affecting blood clotting. Current treatments have limitations, so the study aimed to produce recombinant protein S using an Escherichia coli expression system as a potential therapy. We designed and constructed an expression vector, successfully cloned the PROS1 gene into the vector 2Bc-t, and transformed it into E. coli strain Origami B (DE3). Through optimization steps, we achieved protein expression, however, N-terminal protein degradation remained a concern potentially due to the absence of glycosylation in the prokaryotic expression system. Protein modeling employed in this study provided valuable insights into the impact of specific genetic mutations on protein S structure and function. Future directions include comprehensive characterization, including functional assays, and comparison to standard protein S–representing a step towards an alternative therapeutic solution for protein S deficiency using synthetic biology in healthcare.

The gene sequence encoding human protein S (NCBI Gene ID: 5627) was retrieved from the NCBI database and optimized for expression in our bacterial expression system. A ribosome binding site (RBS) sequence was incorporated , then the optimized sequence was synthesized as gBlocks by Integrated DNA Technologies (IDT). Primers were designed, then used in PCR amplification to flank the PROS1 gene, generating overhangs that were complementary to the LIC site of the vector 2Bc-t. Complete sequences of the constructed gene fragments can be found in the iGEM BioBrick Registry (Part:BBa_K4235011). To confirm the successful insertion of the PROS1 insert into the 2Bc-T plasmid, restriction digestion was performed using XhoI and BamHI restriction enzymes. The presence of the cloned insert was verified by amplifying it from the constructed plasmid using T7 forward and reverse primers. To analyze the protein expression levels, post-induction samples were collected for Western blot analysis. Homology modeling using ChimeraX software was done based on the 1Z6C protein model obtained from the Protein Data Bank ((PDB_1Z6C). The model covered amino acids 200 to 286, an 87 amino acid long sequence verified from solution using NMR.

 Through PCR and Ligation Independent Cloning (LIC), we obtained the protein S gene’s amplicon and cloned it into the 2Bc-T vector. Gel electrophoresis and restriction digestion confirmed the presence of the protein S gene in the recombinant vector, and DNA sequencing validated its presence. Subsequently, we conducted expression tests using E. coli strains BL21 and Origami B to assess protein production. While no expression was observed in BL21 samples, western blot analysis of Origami B samples revealed bands of incorrect sizes, indicating protein degradation. To optimize expression, we induced Origami B cells at lower temperatures and reduced the concentration of IPTG. Western blot analysis of the optimized conditions showed the detection of full-length soluble protein S bands (~70 kD), indicating successful expression. However, degradation of the protein from the N-terminal region was observed, likely due to the absence of glycosylation in the prokaryotic system. These findings highlight the need for further exploration of glycosylation strategies to improve protein stability. Further refinement and characterization are necessary to improve the expression system and ensure the functional similarity of the produced protein S to its human counterpart. The study's findings contribute to the field of synthetic biology and hold promise for future therapeutic solutions for protein S deficiency.