Production of low-expressing recombinant cationic biopolymers with high purity

- Cationic biopolymers with increasing number of cationic residues ranging from 22 to 96 were genetically engineered.
- As the number of cationic residues increased the yield of production decreased.
- The major contaminant during the purification process proved to be SlyD endogenous E. coli protein.
- TALON cobalt based resin helped reduce the impurity during the purification process but did not eliminate completely.
- BL21(DE3)LOBSTR allowed production of all cationic biopolymers with above 99% purity in a single-step purification process.

The growing complexity of recombinant biopolymers for delivery of bioactive agents requires the ability to control the biomaterial structure with high degree of precision. Genetic engineering techniques have provided this opportunity to synthesize biomaterials in an organism such as E. coli with full control over their lengths and sequences. One class of such biopolymers is recombinant cationic biopolymers with applications in gene delivery, regenerative medicine and variety of other biomedical applications. Unfortunately, due to their highly cationic nature and complex structure, their production in E. coli expression system is marred by low expression yield which in turn complicates the possibility of obtaining pure biopolymer. SlyD and ArnA endogenous E. coli proteins are considered the major culprits that copurify with the low-expressing biopolymers during the metal affinity chromatography. Here, we compared the impact of different parameters such as the choice of expression hosts as well as metal affinity columns in order to identify the most effective approach in obtaining highly pure recombinant cationic biopolymers with acceptable yield. The results of this study showed that by using E. coli BL21(DE3) LOBSTR strain and in combination with our developed stringent expression and Ni-NTA purification protocols highly pure products in one purification step (>99% purity) can be obtained. This approach could be applied to the production of other complex and potentially toxic biopolymers with wide range of applications in biomedicine.