Recombinant Human Calcitonin (rHCT) Engineering Service

Recombinant Human Calcitonin (rHCT) is a small peptide hormone primarily used to treat hypercalcemia, Paget's disease, and postmenopausal osteoporosis. Production is highly challenging: it contains a crucial intramolecular disulfide bond and a C-terminal amide group essential for activity. Expression in prokaryotes, such as E. coli, typically results in inactivity in prokaryotic expression due to misfolding and lack of correct disulfide bond pairing in the reducing cytoplasm. Conversely, chemical synthesis is pure but suffers from high cost in chemical synthesis due to the peptide's length (32 amino acids) and complex modifications. Biosynthesis in E. coli combined with refolding remains the most economically viable path, provided folding and purity issues are solved.

CD Biosynsis offers a synthetic biology service focused on high-yield, active rHCT production. Our core strategy involves modification of Escherichia coli fusion expression system , utilizing a high-solubility partner (GST or Trx) to maximize the expression of the Calcitonin precursor while directing the product toward inclusion bodies (for high concentration) or the oxidizing cytoplasm (for in vivo folding). This is coupled with the optimization of correct disulfide bond pairing through controlled in vitro refolding protocols and/or engineering the E. coli host (TrxB/Gor mutations) to create an oxidizing cytoplasmic environment conducive to native folding. This integrated approach aims to deliver high yields of the fully active rHCT peptide, offering a cost-effective alternative to chemical synthesis.

Pain Points

Producing high-quality rHCT efficiently faces these key technical challenges:

• Inactivity in Prokaryotic Expression: The E. coli cytoplasm is highly reducing, preventing the formation of the essential Cys 1 - Cys 7 disulfide bridge , leading to inactive, misfolded product.
• High Cost in Chemical Synthesis: The 32-amino acid chain length and the necessary post-translational modification (C-terminal amidation) make Solid Phase Peptide Synthesis (SPPS) labor-intensive and expensive at industrial scale.
• Refolding Inefficiency: Crude refolding attempts of inclusion bodies often lead to incorrect disulfide bond pairing (non-native isomers) or aggregation, reducing the yield of the active monomer.
• C-Terminal Amidation: Calcitonin requires a C-terminal amide group (-NH2) for full biological activity, a modification E. coli cannot perform natively , necessitating an enzymatic or chemical post-processing step.

A cost-effective solution requires maximizing expression and optimizing the formation of the single, crucial disulfide bond.

Solutions

CD Biosynsis utilizes advanced expression and refolding engineering to optimize active rHCT production in E. coli:

Modification of E. coli Fusion Expression System

           

We select high-solubility tags (NusA, Trx, Sumo) to enhance precursor expression and prevent degradation , followed by an efficient cleavage site (e.g., TEV or Factor Xa).

Optimization of Correct Disulfide Bond Pairing

We develop optimized in vitro refolding protocols (buffer composition, pH, redox agents) to favor the Cys1-Cys7 pairing, maximizing the yield of the native monomer.

Oxidizing Cytoplasm Strain Engineering

We use engineered E. coli strains (e.g., Rosetta-gami or SHuffle cells with Gor/TrxB mutations) that allow the disulfide bond to form correctly in vivo in the cytoplasm.

Enzymatic Amidation Strategy

We integrate a post-purification enzymatic amidation step (using Peptidylglycine alpha-Amiding Monooxygenase) to correctly introduce the essential C-terminal amide group.

This systematic approach is focused on leveraging the cost benefits of E. coli while achieving the structural fidelity required for biological activity.

Advantages

Our rHCT engineering service is dedicated to pursuing the following production goals:

High Yield and Expression Level

Fusion tags and strong promoters ensure maximal expression of the Calcitonin precursor in the E. coli system.

Correct Structural Fidelity

Optimized refolding or in vivo oxidation ensures correct Cys1-Cys7 disulfide bridge formation , resulting in a biologically active peptide.

Cost-Effective Bioproduction

E. coli fermentation is far more scalable and cheaper than multi-step SPPS for large-volume peptide drugs. [Image of Cost Reduction Icon]

High Specific Activity

Inclusion of the post-purification amidation step ensures the essential C-terminal modification for full receptor binding and potency.

Simplified Purification

The fusion tag allows for an efficient one-step initial purification via affinity chromatography before cleavage.

We provide a specialized platform aimed at minimizing the cost and maximizing the quality of clinically viable rHCT production.

Process

Our rHCT strain engineering service follows a rigorous, multi-stage research workflow:

• Fusion Construct Design: Design the Calcitonin gene fused to a high-solubility tag and a specific protease cleavage site , codon-optimized for E. coli.
• Expression and Inclusion Body Recovery: Express the fusion protein under strong induction and optimize lysis/wash steps to recover high-purity inclusion bodies.
• Refolding Optimization: Screen and optimize pH, temperature, and the ratio of oxidizing/reducing agents (e.g., GSSG/GSH) to maximize the yield of the correctly folded monomer.
• Cleavage and Amidation: Perform enzymatic or chemical cleavage to remove the fusion tag, followed by enzymatic amidation of the C-terminus.
• Final Purification and Quality Control: Use Reverse-Phase HPLC (RP-HPLC) to purify the active rHCT and confirm purity and structure via Mass Spectrometry and CD spectroscopy.
• Result Report Output: Compile a detailed Experimental Report including strain data, refolding protocols, and final titer/quality metrics , supporting regulatory and scale-up efforts.

Technical communication is maintained throughout the process, focusing on timely feedback regarding folding efficiency and product integrity.

Explore the potential for a high-quality, cost-effective rHCT supply. CD Biosynsis provides customized peptide production solutions:

• Detailed Refolding Yield and Purity Analysis Report , demonstrating the success of the disulfide bond formation optimization.
• Consultation on enzymatic amidation implementation and C-terminal modification efficiency.
• Experimental reports include complete raw data on final active rHCT titer (mg/L) and structural homogeneity , essential for clinical development.