Zdenko Levarski

High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli

Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

Purification of small-size acidic proteoglycan-like domain of carbonic anhydrase IX fused with thioredoxine expressed in Escherichia coli for structural characterization

Abstract Proteoglycan-like (PG) domain of carbonic anhydrase IX (CA IX) is a small-sized acidic domain (8.3 kDa; pI ∼ 3.5) with a very unusual amino acid composition. More than one third of its sequence consists of acidic amino acids and it contains no aromatic residues. It has been revealed that it holds one of the key roles in oncogenetic mechanisms, in which CA IX participates. However, it has not been structurally characterized yet. With these prospects, we developed an expression system of recombinant PG domain production in the form of a fusion protein using thioredoxine (Trx) as the fusion partner in Escherichia coli. We performed three steps during purification process utilizing affinity chromatography, anion-exchange chromatography and gel filtration; and the highest purity we reached was 93.7% with protein concentration 23.3 mg/L. Hypothetical yield of the recombinant protein would be 0.581 mg/L of culture. The applicability of the fusion protein Trx-PG for further structural studies was confirmed by in silico structure prediction tool and ELISA.