Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.
Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.
Kushnir
Susanna
S
Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Marsac
Yoann
Y
Breitling
Reinhard
R
Granovsky
Igor
I
Brok-Volchanskaya
Vera
V
Goody
Roger S
RS
Becker
Christian F W
CF
Alexandrov
Kirill
K
D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004952:Esters / Q000737:chemistry; D019793:Fluorescein / Q000737:chemistry; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004952:Esters; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D001710:Biotin; D020691:rab GTP-Binding Proteins; D003545:Cysteine; D019793:Fluorescein