3.1 Choose Your Protein

For this assignment, I have chosen the HindIII restriction enzyme. HindIII is a type II restriction endonuclease that recognizes the palindromic sequence AAGCTT and cleaves it between the two adenines. This enzyme is widely used in molecular biology for DNA manipulation, cloning, and genetic engineering.

Protein sequence HindII from UniProt:

3.2. Reverse Translation: Protein to DNA

Using a reverse translation tool, I inferred the possible nucleotide sequence corresponding to the HindIII protein. Since multiple codons can encode the same amino acid, there are various possible DNA sequences that could translate into this protein.

Reverse translation protein to ADN

3.3. Codon Optimization

Codon optimization is a crucial step in recombinant protein expression. Different organisms have distinct codon usage preferences due to variations in tRNA availability. By optimizing the codons, we can enhance translation efficiency, increase protein yield, and minimize translational errors.

For this project, I optimized the nucleotide sequence of the HindIII protein for Escherichia coli (E. coli). This bacterium is widely used as a host for recombinant protein production due to its rapid growth, well-characterized genetics, and cost-effectiveness. The optimized sequence was generated using an online codon optimization tool, ensuring higher expression efficiency in E. coli.

Optimized DNA sequence for E. coli is as follows

3.4. You Have a Sequence! Now What?

Once the DNA sequence is optimized, it can be used to express the HindIII protein using different biotechnological approaches. The two main methods to produce recombinant proteins are:

1. Cell-Dependent Expression (E. coli Expression System)

• The optimized DNA sequence can be cloned into an expression vector (e.g., pET, pUC).
• The plasmid is then transformed into E. coli competent cells.
• After induction with IPTG or another suitable inducer, the bacteria transcribe and translate the DNA into the HindIII protein.