title: mRNA secondary structure engineering of Thermobifida fusca endoglucanase (Cel6A) for enhanced expression in Escherichia coli.
abstract: The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5'-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the 5'-end of mRNA to relieve hybridization with ribosomal binding site. Two variants of glycoside hydrolase family six endoglucanase, wild type (cel6A.wt) and mutant (cel6A.mut) from Thermobifida fusca were expressed and characterized in E. coli using T7 promoter-based expression vector; pET22b(+). Enhanced expression level of engineered construct (Cel6A.mut) with ∆G = -2.7 kcal mol(-1)was observed. It showed up to ~45 % higher expression as compared to the wild type construct (Cel6A.wt) having ∆G = -7.8 kcal mol(-1) and ~25 % expression to the total cell proteins. Heterologous protein was purified by heating the recombinant E. coli BL21 (DE3) CodonPlus at 60 °C. The optimum pH for enzyme activity was six and optimum temperature was 60 °C. Maximum activity was observed 4.5 Umg(-1) on CMC. Hydrolytic activity was also observed on insoluble substrates, i.e. RAC (2.8 Umg(-1)), alkali treated bagass (1.7 Umg(-1)), filter paper (1.2 Umg(-1)) and BMCC (0.3 Umg(-1)). Metal ions affect endoglucanase activity in different ways. Only Fe(2+) exhibited 20.8 % stimulatory effects on enzyme activity. Enzyme activity was profoundly inhibited by Hg2(+) (91.8 %).
authors.affiliation: Department of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur, 10250, AJK, Pakistan, aliimran@must.edu.pk.
authors.forename: Imran
authors.initials: I
authors.lastname: Ali
authors.forename: Rehana
authors.initials: R
authors.lastname: Asghar
authors.forename: Sajjad
authors.initials: S
authors.lastname: Ahmed
authors.forename: Muhammad
authors.initials: M
authors.lastname: Sajjad
authors.forename: Muhammad
authors.initials: M
authors.lastname: Tariq
authors.forename: M
authors.initials: M
authors.lastname: Waheed Akhtar
mesh_terms: D039903:Actinobacteria / Q000201:enzymology*; D017123:Bacteriophage T7 / Q000235:genetics; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002480:Cellulase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002482:Cellulose / Q000378:metabolism; D020536:Enzyme Activators / Q000032:analysis; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D060847:Metabolic Engineering / Q000379:methods; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics*; D012335:RNA, Ribosomal / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D011994:Recombinant Proteins; D002482:Cellulose; C027433:bagasse; D002480:Cellulase