Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli
Journal of the Serbian Chemical Society
Herein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum) aspartic protease (FeAPL1) in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion partner. His 6 -FeAPL1 was produced in large quantities as an insoluble protein localized in inclusion bodies. On the other hand, MBP-FeAPL1 was localized in both the cytoplasm and inclusion bodies in
... inclusion bodies in BL21 and Rosetta-gami strains. Only purified soluble MBP-FeAPL1 from Rosetta-gami cells showed proteolytic activity at pH 3.0 with BSA as the substrate. The results also indicated that FeAPL1 contained a PRO segment that had to be removed for the enzyme activity to appear. The activity of FeAPL1 produced in the Rosetta-gami strain, which enables disulfide bond formation, indicated the importance of the twelve cysteine residues for correct folding and functionality. 608 MILISAVLJEVIĆ et al. wheat seed cDNA library. Analysis of the polypeptide deduced from the FeAPL1 coding region, predicted an M w of 48.6 kDa, four N-glycosylation sites and a hydrophobic signal peptide in the N-terminal region. Active-site sequence motifs DTG/DSG characteristic for APs as well as twelve Cys residues were also registered. 7 Interestingly, bioinformatics analysis of the Arabidopsis genome sequence revealed 59 AP-like proteins, providing a new perspective concerning the diversity of AP family members in plants. 8 The biological significance of the existence of two types of APs only in plants is not clear. Little is known about the biological functions and biochemical properties of the AP-like members. Various functions have been proposed. It was reported that they could be involved in pathogen resistance, 4 in the degradation of rubisco during leaf senescence, 6 in prey digestion 5 or in nucellar cell death. 2, 3 One reason for the lack of data related to the AP-like group is that it is very difficult to obtain sufficiently purified enzyme from plant tissues for detailed characterization. One suitable way is to overexpress the gene in heterologous systems, such as microorganisms or cell cultures of higher organisms (yeast, insects, mammals, etc.). The most common host for the production of recombinant APs is Escherichia coli. However, of the many attempts, those that gave successful expression often yielded a completely insoluble product that had to be refolded to gain activity. This occurred with OsAsp1 from rice, 3 deleted forms of CND41 from tobacco, 9 cardosin A from Cynara cardunculus 10 and some non-plant APs--candidapepsin from Candida tropicalis, 11 bovine prochymosin, 12 porcine pepsin 13 and human procathepsin D. 14 The successful production of plant APs in higher organisms was reported for cyprosin and phytepsin in Pichia pastoris 15 and insect cells, 16 respectively. The attractiveness of E. coli as an expression system lies in its ability to grow rapidly and at high density on inexpensive substrates, its well characterized genetics and the availability of an increasingly large number of cloning vectors and mutant host strains. 17 The most important factors that largely affect efficient recombinant protein expression are: a) a strong and tightly regulated promoter (isopropyl--D-thiogalactopyranoside (IPTG)-inducible promoters are mostly used); b) E. coli strains deficient in the most harmful proteases and/or thioredoxin and glutathione reductases; c) codon usage difference between the E. coli strain and the overexpressed gene; d) solubility of the recombinant protein, which depends on the protein expression rate, presence of disulfide bonds, hydrophobicity and choice of fusion partner (tags). 18 The most commonly used tags are polyhistidine (His tag) and glutathione S-transferase (GST tag), but tags such as thioredoxin, MBP (Maltose Binding Protein), NusA (transcription termination anti-terminator factor) 19 or SUMO (small ubiquitin-like modifier) 20 have been shown to be more effective as solubility enhancers of fusion partners. For inso-617 CONCLUSIONS Expression of recombinant proteins is a challenging process that demands empirical investigations of all expression system actors, as it is usually impossible to predict the behavior and features of the recombinant protein. Therefore, there are no "hard proteins" if all actors in the expression system are analyzed. These investigations should lead toward obtaining a satisfactory amount of soluble and biologically active protein. In the case of FeAPL1, the less reducing cytoplasm of the Rosetta gami strain in combination with the MBP tag with solubilizing properties on its fusion partner, enabled the production of a satisfactory amount of soluble and, more importantly, proteolytically active enzyme.