Best Basic Part

α-S1-casein (Bos taurus)
with N-terminal 6xHisTag and TEV-site

The part contains a gene CSN1S1 coding for Bos taurus a-S1-casein or CASA1_BOVIN. Located on chromosome 6, the total length of the gene itself is 642 nt, coding for 214 amino acid long sequence.

Protein constituting amino acid sequence was obtained from UniProt [1] and reverse translated to the nucleotide sequence, which was then optimized for expression in E. coli via IDT Codon Optimization Tool.

Link to the registry: registry.igem.org/parts/bba-25orz956 https://www.uniprot.org/uniprotkb/P02662/entry#sequences

CASA1_BOVIN

Casein is a protein found in milk, and out of four types of casein proteins – alpha S1-casein, alpha S2-casein, beta-casein, and kappa-casein, alpha-S1-casein is the predominant protein in bovine milk, accounting for about 31% of the total milk proteins and up to 40% of the total casein fraction. α-S1-casein plays a fundamental role in the capacity of milk to transport calcium phosphate [2]. CSN1S1 gene is highly polymorphic due to single nucleotide polymorphisms, insertions and deletions. These polymorphisms are associated with milk yield, protein content and its coagulation properties. Besides being a constituent protein of the milk, proteolytic cleavage of a-S1-casein results in the formation of casokinins – bioactive peptides with antihypertensive properties [3]. Several studies report antimicrobial and antioxidant properties of a-s1-casein, which could potentially be valuable in developing additives for the food industry and medical applications [4]. However, it is well known that bovine a-S1-casein is an allergen, responsible for IgE-mediated allergic reactions. Multiple IgE binding epitopes have been identified in bovine a-S1-casein [5], which highlights the necessity of caution when developing applications for medicinal or dietary use. Casein materials have previously been explored for their potential use in wound healing applications [6], which prompted us to deeper explore the possibilities of using milk proteins in various burn wound healing approaches.

CASA1_BOVIN

In our part, the N-terminal 6xHisTag was introduced into the protein sequence after the Start codon. The addition of affinity tags such as the 6xHisTag is a common strategy in protein engineering to facilitate purification, detection, and stabilisation of recombinant proteins. 6xHisTag allows purification by Ni2+ affinity chromatography. HisTag can be added to either the N-terminal or C-terminal end of a protein. One of the considerations for choosing the N-terminal end of the 6xHisTag insertion in our construct was the fact that caseins are intrinsically disordered proteins and the N-terminal end is more accessible than the C-terminus (https://doi.org/10.3389/fvets.2022.952319) and shows greater thermostability and structural integrity (https://doi.org/10.3168/jds.S0022-0302(05)72910-6). In that way there is a higher chance that insertion of a 6xHisTag sequence will not significantly alter protein structure after protein expression; thus, the 6xHisTag will be more accessible for protein purification if inserted in the N-terminal end of the protein. Besides that, N-terminal fusions are the most common and often enhance soluble protein expression (DOI:10.1002/0471140864.ps0524s61.

N-terminal positioning of 6xHisTag allows for a convenient insertion of a following TEV protease recognition site. TEV protease cleavage is often used for tag removal from recombinant proteins, as it recognises a highly specific sequence ENLYFQ↓S, ensuring precise cleavage. Insertion of the recognition site at the N-terminus guarantees that only the serine residue is left after the cleavage, which restores the protein to its nearly native form (doi: 10.1007/978-1-4939-6887-9_14).

Described modifications were added to the gene construct in silico, and gene synthesis was obtained from GenScript. The gene construct was cloned into the pET-24a(+) vector, notable for its successful usage for protein production owing to the T7 expression system regulated by IPTG induction that it operates on.

The received gene fragment was amplified by PCR using Phusion polymerase; successful amplification was confirmed by NAGE. The modified gene fragment was then digested by XhoI and NdeI FastDigest restriction enzymes, as well as the pET-24a(+) plasmid. Afterwards, ligation was performed using T4 DNA ligase. Fig. 1

Further E. coli DH5-α cells were transformed with the resulting plasmid construct for plasmid amplification and following purification. The transformation was successful. Fig. 2

For protein production, it was initially decided to use E. coli BL21(DE3) strain, as it is one of the standard and most widely used strains for recombinant protein production [10]. For that, competent BL21(DE3) cells were transformed with the α-S1-casein gene containing plasmid construct. The transformation was successful, and bacterial colonies containing the plasmid were further used for protein expression.

Transformed BL21(DE3) were cultivated in 2TY media with kanamycin until the OD600 reached 0.6 - 0.9, and then protein production was induced by adding 1M IPTG to the final concentration of 1 mM. Protein expression was continued at 37 C for 3 hours, after which cells were harvested by centrifugation.

Cells were then lysed by ultrasonification, and the whole lysate, as well as supernatant (soluble protein fraction) and pellet (insoluble protein fraction), were analysed by SDS-PAGE gel electrophoresis. Fig. 2a

Protein expression was also carried out according to overnight protein expression protocol at 20°C and lower final IPTG induction concentration (0.5 mM). Lowering expression temperature and IPTG concentration at induction reportedly improves protein production yield and offers a higher chance of soluble protein production [11]. Fig. 2b

To optimise soluble protein production, it was decided to use an alternative E. coli strain – Rosetta 2, which is used to enhance production of eukaryotic proteins in E. coli. Protein expression was carried out by the same 20 °C overnight protein production protocol described for BL21(DE3). Fig. 3

The α-S1-casein protein appeared to be only partly insoluble, which would enable the purification of soluble fraction by HisTrap Ni²⁺ affinity chromatography. The Rosetta 2 cells from protein expression at 20 °C overnight were lysed by ultrasonification, lysate was clarified by centrifugation and loaded onto HisTrap FF affinity column. SDS-PAGE analysis of the eluate fractions shows that protein indeed contains a functional 6xHisTag which allows purification via Ni²⁺ affinity chromatography. Fig. 4

In conclusion, we have created a part which, upon insertion into a pET-24a(+) expression vector, allows for the production of α-S1-casein of bovine origin. The protein is partly soluble and some fraction of it is possible to purify using Ni²⁺ affinity chromatography.