A rapid solubility-optimized screening procedure for recombinant subtilisins in E. coli

Abstract

Subtilisins and other serine proteases are extensively used in the detergent, leather and food industry, and frequently under non-physiological conditions. New proteases with improved performance at extreme temperatures and in the presence of chemical additives may have great economical potential. The increasing availability of genetic sequences from different environments makes homology-based screening an attractive strategy for discovery of new proteases. A prerequisite for large-scale screening of protease-encoding sequences is an efficient screening procedure. We have developed and implemented a screening procedure that encompasses cloning of candidate sequences into multiple expression vectors, cytoplasmic expression in E. coli, and a casein-based functional screen. The procedure is plate-format compatible and can be completed in only four days, starting from the gene of interest in a suitable cloning vector. The expression vector suite includes six vectors with combinations of maltose-binding protein (MBP) or the small ubiquitin-related modifier (SUMO) for increased solubility, and polyhistidine tags for downstream purification. We used enhanced green fluorescent protein and four Bacilli subtilisins to validate the screening procedure and our results show that proteins were expressed, soluble and active. Interestingly, the highest activities were consistently achieved with either MBP or SUMO fusions, thus demonstrating the merit of including solubility tags. In conclusion, the results demonstrate that our approach can be used to efficiently screen for new subtilisins, and suggest that the approach may also be used to screen for proteins with other activities.

Mutational analysis of the pro‐peptide of a marine intracellular subtilisin protease supports its role in inhibition

“…To facilitate enzyme expression we used our previously developed screening procedure for subtilisin‐like serine proteases . The Planococcus sp.…”

“…The isp gene was sub‐cloned from the delivery vector to a suite of expression vectors using a fragment exchange (FX) cloning method . Construction of the expression vectors have been described previously …”

“…Primers in Supporting Information Table S2 were used to amplify the truncated ISP versions by PCR using Phusion polymerase. Gene fragments were purified, and cloned into the pINITIAL cloning vector by FX‐cloning . Plasmids were sequenced to confirm correct truncations.…”

“…Small‐scale recombinant expression was carried out according to the protocol described previously in 4 mL culture volumes. Following expression, cells were collected and resuspended in 1 mL lysis buffer (50 m M Tris‐HCl pH 8.5, 50 m M NaCl, 0.25 mg/mL lysozyme, 10% (v/v) glycerol).…”

Insight into subtilisin E-S7 cleavage pattern based on crystal structure and hydrolysates peptide analysis

“…Thus, Subtilisin E is typically obtained in inclusion body and purified through a complicated renaturation process [23-25] and the highest yield was estimated to be 40-50 mg/liter of culture[26]. Recently, Gro Elin et al overcame this problem by fusing maltose-binding protein (MBP) or the small ubiquitin-related modifier (SUMO) to Subtilisins[27]. But we found that SES7 is soluble without such modifications (Figure S2).…”