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A method for the transient inhibition of toxicity of secretory recombinant proteins, exemplified by bacterial alkaline phosphatase. Novel protocol for problematic DNA termini dephosphorylation.

ABSTRACT: Genes encoding proteins 'toxic' to recombinant host are difficult for cloning/expression and recombinant clones are unstable. Even tightly controlled inducible T7-lac, P_BAD, P_L, P_R promoters are not totally silent in an uninduced state and thus not adequate for highly toxic proteins. An innovative approach to engineering and expression of the gene, encoding bacterial alkaline phosphatase (BAP) is proposed. The native precursor enzyme contains a signal peptide at the N-terminus and is secreted to the Escherichia coli (E. coli) periplasm. The signal peptide is then removed that allows oxidation and formation of active dimers. To decrease toxicity of the bap gene, its secretion leader coding section was replaced with a N-terminal His6-tag. The gene was expressed in E. coli in a P_BAD vector, resulting in the accumulation of soluble His6-BAP in the cytoplasm. The His6-BAP was neutral to the cells, as no maturation was possible in the reducing cytoplasm. The purified homogenous protein was further reactivated in a redox buffer containing the protein structure stabilizing cofactors. The His6-BAP exhibited high activity. A dephosphorylation protocol for all types of DNA termini was developed.The method appears well suited for the industrial production of BAP and can be applied to other problematic proteins.• Efficient toxic gene expression • Novel approach to toxic gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme • Scaled-up production of ultrapure BAP • Improved protocol for all types of DNA termini dephosphorylation.

SUBMITTER: Krawczun N

PROVIDER: S-EPMC8374387 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:The biotechnology production of enzymes is often troubled by the toxicity of the recombinant products of cloned and expressed genes, which interferes with the recombinant hosts' metabolism. Various approaches have been taken to overcome these limitations, exemplified by tight control of recombinant genes or secretion of recombinant proteins. An industrial approach to protein production demands maximum possible yields of biosynthesized proteins, balanced with the recombinant host's viability. Bacterial alkaline phosphatase (BAP) from Escherichia coli (E. coli) is a key enzyme used in protein/antibody detection and molecular cloning. As it removes terminal phosphate from DNA, RNA and deoxyribonucleoside triphosphates, it is used to lower self-ligated vectors' background. The precursor enzyme contains a signal peptide at the N-terminus and is secreted to the E. coli periplasm. Then, the leader is clipped off and dimers are formed upon oxidation. RESULTS:We present a novel approach to phoA gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme. The recombinant bap gene was modified by replacing a secretion leader coding section with a N-terminal His6-tag, cloned and expressed in E. coli in a PBAD promoter expression vector. The gene expression was robust, resulting in accumulation of His6-BAP in the cytoplasm, exceeding 50% of total cellular proteins. The His6-BAP protein was harmless to the cells, as its natural toxicity was inhibited by the reducing environment within the E. coli cytoplasm, preventing formation of the active enzyme. A simple protocol based on precipitation and immobilized metal affinity chromatography (IMAC) purification yielded homogeneous protein, which was reactivated by dialysis into a redox buffer containing reduced and oxidized sulfhydryl group compounds, as well as the protein structure stabilizing cofactors Zn2+, Mg2+ and phosphate. The reconstituted His6-BAP exhibited high activity and was used to develop an efficient protocol for all types of DNA termini, including problematic ones (blunt, 3'-protruding). CONCLUSIONS:The developed method appears well suited for the industrial production of ultrapure BAP. Further, the method of transient inactivation of secreted toxic enzymes by conducting their biosynthesis in an inactive state in the cytoplasm, followed by in vitro reactivation, can be generally applied to other problematic proteins.

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A method for the transient inhibition of toxicity of secretory recombinant proteins, exemplified by bacterial alkaline phosphatase. Novel protocol for problematic DNA termini dephosphorylation.

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