title: A novel method for increasing the expression level of recombinant proteins.
abstract: Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.
authors.affiliation: Department of Research and Development, Corixa Corporation, 1124 Columbia Street, Seattle, WA 98104, USA. wanga@corixa.com
authors.forename: Aijun
authors.initials: A
authors.lastname: Wang
authors.forename: Jonathan
authors.initials: J
authors.lastname: Clapper
authors.forename: Jeffery A
authors.initials: JA
authors.lastname: Guderian
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Foy
authors.forename: Gary R
authors.initials: GR
authors.lastname: Fanger
authors.forename: Marc W
authors.initials: MW
authors.lastname: Retter
authors.forename: Yasir A W
authors.initials: YA
authors.lastname: Skeiky
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D004926:Escherichia coli; D005434:Flow Cytometry; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D000942:Antigens, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins