A novel concept of Pyridoxal 5'-phosphate permeability in E.coli for modulating the heterologous expression of PLP dependent proteins. (February 2022)

• Record Type: Journal Article
• Authors: Saxena, Vijay Kumar; Vedamurthy, G.V.; Singh, Raghvendar
• Is Part Of: Process biochemistry. Volume 113(2022)
• Page Start: 37
• Page End: 46
• Publication Date: 2022-02
• Subjects: PLP Pyridoxal 5′-phosphate -- PL Pyridoxal -- PM Pyridoxamine -- PN Pyridoxine -- OASS O-acetylserine sulphdrylase -- DXP Deoxyxylulose 5-phosphate -- CDS Coding sequence; Pyridoxal 5'-phosphate -- PLP -- Protein expression -- Cofactor -- Escherichia coli -- Fluorescence quenching; Biochemical engineering -- Periodicals; Biotechnology -- Periodicals; Biochemistry -- periodicals; Biotechnology -- periodicals; Chemical Engineering -- periodicals; Génie biochimique -- Périodiques; Biotechnologie -- Périodiques; Biochemical engineering; Biotechnology; Periodicals; 660.63
• DOI: 10.1016/j.procbio.2021.12.016
• Languages: English
• ISSNs: 1359-5113

Abstract

Graphical abstract: Highlights: A novel concept of demonstrated pyridoxal phosphate permeability in E.coli Pyridoxal phosphate inclusion in media increases protein yield and enzymatic activity A novel protein model system for pyridoxal phosphate linked permeation studies. Abstract: PLP (Pyridoxal 5′-phosphate) dependent proteins are important drug targets and effector molecules, and thus, their heterologous overexpression is of industrial importance and has commercial value. We have predicted the docking site of PLP on O-acetylserine sulphdrylase (OASS) of H.contortus and determined that the lysine-47 is very important for the binding of PLP. We have used this protein as a model protein for testing the effect of PLP on the expression of PLP dependent proteins by E.coli . Soluble recombinant protein could be purified from each of the culture vials grown with variable amount of PLP [0 mM (Group I), 0.01 mM (Group II), 0.025 mM (Group III), 0.05 mM (Group IV) and 0.1 mM (Group V)]. There was approximately 4.2 %, 7.2 %, 10.5 % and 18 % increase in purified protein yield in Group II, III, IV and V, respectively, in comparison to group I. There was a significant quenching of tryptophan fluorescence emission in groups II, III, IV and V compared to group I. We could find a PLP concentration-dependent increase in expression and catalytic activity signifying the probable transport of PLP across the membrane. E.coli does not secrete any dephosphorylation factor for PLP in the immediate microenvironment. There was a significant reduction in levels of inorganic phosphate after PLP dephosphorylation in induced group in comparison to uninduced E.coli and BL21 groups, signifying PLP internalization in E.coli . The mechanism of transport of PLP in the light of the current experiment is still unknown and should be a subject of future studies.