Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli
One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. N-carbamoyl-D-amino-acid amidohydrolase (DCase) is an important enzyme involved in semi-synthesis of β-lactam antibiotics in industry. In this study, in order to determine the amino acid sites responsible for solubility in DCase, error-prone PCR and DNA shuffling techniques are applied to randomly mutate its encoding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation are isolated. Solubility tests of these mutants and several other mutants generated by site-directed mutagenesis indicate that three amino acid residues of DCase (A18, Y30 and K34) are related to the protein solubility in DCase. In silico structural modeling analyses further suggest that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on the information, multiple engineering designated mutants were constructed by site-directed mutagenesis; among them, a triple mutant A18T/Y30N/K34E (named as DCase-M3) can be over-expressed in E. coli with up to 80% of DCase-M3 protein as soluble. DCase-M3 is purified to homogeneity and a comparative analysis with WT DCase demonstrates that DCase-M3 enzyme is similar to the native DCase in terms of its kinetic and thermodynamic properties. The study provides new insights on recombinant protein solubility in E. coli.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 912715
- Report Number(s):
- PNNL-SA-53029; BIJOAK; TRN: US200801%%1159
- Journal Information:
- Biochemical Journal, 402:429-437, Vol. 402; ISSN 0006-2936
- Country of Publication:
- United States
- Language:
- English
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