The step that proved to be the main challenging obstacle in the production of human insulin by genetic engineering is :

Options

A. Removal of C-peptide from active insulin
B. Getting insulin assembled into a mature form
C. Addition of C-peptide to proinsulin
D. Splitting A and B polypeptide chains

Answer

To solve the question regarding the main challenging obstacle in the production of human insulin by genetic engineering, we can break down the process step by step.

### Step-by-Step Solution:

1. **Understanding Pro-insulin Structure**:
- Human pro-insulin consists of three peptide chains: A, B, and C. The C-peptide is present in the immature form of insulin.

2. **Maturation Process**:
- During the maturation of pro-insulin into active insulin, the C-peptide is removed. The active form of insulin is composed only of the A and B chains.

3. **Genetic Engineering Process**:
- In genetic engineering, the gene coding for insulin is inserted into a plasmid of E. coli bacteria. This allows the bacteria to produce insulin chains A and B.

4. **Production of Insulin Chains**:
- The E. coli bacteria are cultured to produce the insulin chains in larger quantities. However, the chains A and B are produced separately.

5. **Combining Insulin Chains**:
- The final step involves combining the A and B chains to form mature insulin. This is done by creating disulfide bonds between the chains.

6. **Main Challenge Identified**:
- The main challenge in the production of human insulin by genetic engineering is the assembly of insulin into its mature form, specifically the removal of the C-peptide and the correct assembly of the A and B chains.

### Conclusion:
The correct answer to the question is **"getting insulin assembled into a mature form"**. This step is crucial because the presence of the C-peptide in pro-insulin must be addressed to produce functional insulin.