title: A new Gateway vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis.
abstract: A major obstacle associated with recombinant protein over-expression in Escherichia coli is the production of insoluble inclusion bodies, a problem particularly pronounced with Mycobacterium tuberculosis proteins. One strategy to overcome the formation of inclusion bodies is to use an expression host that is more closely related to the organism from which the proteins are derived. Here we describe methods for efficiently identifying M. tuberculosis proteins that express in soluble form in Mycobacterium smegmatis. We have adapted the M. smegmatis expression vector pYUB1049 to the Gateway cloning system by the addition of att recombination recognition sequences. The resulting vector, designated pDESTsmg, is compatible with our in-house Gateway methods for E. coli expression. A target can be subcloned into pDESTsmg by a simple LR reaction using an entry clone generated for E. coli expression, removing the need to design new primers and re-clone target DNA. Proteins are expressed by culturing the M. smegmatis strain mc(2)4517 in autoinduction media supplemented with Tween 80. The media used are the same as those used for expression of proteins in E. coli, simplifying and reducing the cost of the switch to an alternative host. The methods have been applied to a set of M. tuberculosis proteins that form inclusion bodies when expressed in E. coli. We found that five of eight of these previously insoluble proteins become soluble when expressed in M. smegmatis, demonstrating that this is an efficient salvage strategy.
authors.affiliation: Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, Thomas Building, 3a Symonds Street, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Rachael M
authors.initials: RM
authors.lastname: Goldstone
authors.forename: Nicole J
authors.initials: NJ
authors.lastname: Moreland
authors.forename: Ghader
authors.initials: G
authors.lastname: Bashiri
authors.forename: Edward N
authors.initials: EN
authors.lastname: Baker
authors.forename: J
authors.initials: J
authors.lastname: Shaun Lott
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D002479:Inclusion Bodies / Q000378:metabolism; D008828:Microbiological Techniques; D020102:Mycobacterium smegmatis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000737:chemistry; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D012995:Solubility; D013997:Time Factors; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D006639:Histidine