title: An improved strategy for easy process monitoring and advanced purification of recombinant proteins.
abstract: In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin-agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.
authors.affiliation: Laboratoire de Biologie Moléculaire, Ecole de Biologie Industrielle, 32 Boulevard du port, 95094, Cergy-Pontoise cedex, France.
authors.forename: Baligh
authors.initials: B
authors.lastname: Miladi
authors.forename: Cyrine
authors.initials: C
authors.lastname: Dridi
authors.forename: Ahmed
authors.initials: A
authors.lastname: El Marjou
authors.forename: Guilhem
authors.initials: G
authors.lastname: Boeuf
authors.forename: Hassib
authors.initials: H
authors.lastname: Bouallagui
authors.forename: Florence
authors.initials: F
authors.lastname: Dufour
authors.forename: Patrick
authors.initials: P
authors.lastname: Di Martino
authors.forename: Abdellatif
authors.initials: A
authors.lastname: Elm'selmi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives; D033921:Vesicular Transport Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; C049538:streptavidin-agarose; C452700:streptavidin-binding peptide; C033223:polyhistidine; D006639:Histidine; D012685:Sepharose; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase