Application of In-Fusion Cloning for the Parallel Construction of E. coli Expression Vectors - DNA Cloning and Assembly Methods - page 212

Fig. 2 Expression of Neisseria meningitidis MC58 putative cell binding factor (NMB0345; amino acids 21-288)
in E. coli using pOPIN vectors. ( a ) The pOPINF family of fusion vectors. SDS-PAGE of expression screen results
from Rosetta2 (DE3) plysS with IPTG induction of Neisseria meningitidis MC58 putative cell binding factor
(NMB0345; amino acids 21-288) fused with members of the pOPINF family shown in Table 1 . The vector is
indicated above the lane and the results are shown in increasing order of size of fusion tag, although as noted
previously the SUMO fusion runs with an anomalously high molecular weight [ 27 ]. ( b ) Fusion at the C-terminal.
SDS-PAGE of expression screen results from Rosetta2 (DE3) plysS with IPTG induction of Neisseria meningiti-
dis MC58 putative cell binding factor (NMB0345; amino acids 21-288) fused with C-terminal fusion vectors
appropriate for expression in the cytoplasm of E. coli shown in Table 2 . The vectors are indicated above the
lane and they are shown in increasing order of size of fusion tag
since they share the same primer extensions. In Fig. 3 we show an
example of the production of two related sub-complexes of
Saccharomyces cerevisiae eIF3 using these vectors [ 35 ]. Each binary
complex was assembled by co-expression of one component cloned
into pOPINF and the other cloned into pOPINRSF. Using the
vectors in Tables 1 , 2 , and 3 up to three proteins may be co-
expressed in a multiple tag format without any deviations from the
standard protocol. It is possible to make polycistronic binary and
ternary expression vectors by In-Fusion™ cloning in the pOPIN