Expression and purification of His-t

fila
A. Mai
Department of Chemistry

ABSTRACT
The SulA division inhibitor of E. coli is repressed by the LexA protein and induced by DNA damage. In
selections for suppression of SulA-mediated filamentation, mutations carrying an IS1 insertion in yqgE
were detected. The yqgE25::IS1 and yqgE27::IS1 mutations also caused slow growth at 37ºC and cold
sensitivity. The yqgE gene is co-transcribed with the downstream gene yqgF, which is essential for
viability. In the yqgE25::IS1 mutant, transcription of yqgF is reduced. Recently, yqgF has been shown to
prevent Rho-dependent transcription termination at intragenic sites.

In complementation experiments between yqgE25::IS1 and pBAD expression vectors carrying yqgE,
yqgF or yqgEF, partial reversal of the cold sensitivity phenotype was observed with yqgF, yqgE had no
effect, but yqgEF completely restored the wild type phenotype. This result suggested that the two gene Figure 2. The pBAD18-Kan::yqgE+ yqg
products work together. To explore the possibility that YqgE and YqgF physically interact, the expression vector.
corresponding coding regions were cloned into His6 expression vectors, and the tagged proteins
purified. Coding sequences were amplified by polymerase chain reaction in preparation for TA cloning.
The stop codon was omitted from the reverse primer for yqgF. The yqgE gene was cloned into pTrcHis,
creating an N-terminal His6 fused sequence, whereas the yqgF sequence was cloned into pTrcHis-2 for
construction of a C-terminal His6 sequence. Plasmids carrying the correct sequences and orientation
with respect to the pTrc promoter were identified. Cells carrying pTrcHis::yqgE or pTrcHis2::yqgF were RESULTS
grown in selective media and expression of the His-tagged proteins induced. The proteins were purified
by NTA-Ni2+ agarose affinity chromatography. Molecular weights determined by SDS-PAGE matched
the expected sizes for the fusion proteins. Western Blotting confirmed the presence of His6-epitope.
Plasmid-mediated co-expression of yqgE+ and yqgF+
Both proteins were relatively stable in crude extracts and after purification, and will be used for in vitro reverses the cold sensitivity caused by a chromosomal
studies designed to detect either strong or weak interactions. To mimic possible in vivo conditions for
YqgE-YqgF interaction, potential co-factors such as DNA, RNA or proteins present in crude extracts yqgE25::IS1 mutation.
will be included in the binding studies. Complementation analysis was performed between yqgE25::IS1 and yqgE+, yqgF+ or yqgE+
cloned into pBAD18 (Figure 2). The pBAD plasmids were constructed in earlier work. The pBA
plasmid, which lacks a Shine Delgarno sequence (ribosome binding site), was used. E. coli
BACKGROUND chromosomal Shine Delgarno sequences upstream of yqgE or yqgF were included in the inserts.
In previous work, a mutant carrying an IS1 insertion in the yqgE gene of E. coli was shown to Plasmids having the desired inserts in the same orientation as the pBAD promoter were identifi
allow division in the presence of the SulA division inhibitor. The sulA gene is one of sequencing. Gene expression from the pBAD promoter was induced by L-arabinose. The cold
approximately 40 “SOS genes” regulated by the LexA repressor and induced by DNA damage sensitivity caused by yqgE25::IS1 is partially reversed by high level expression of yqgF +and
(Figure 1). Recently, yqgF has been shown to prevent Rho-dependent transcription termination completely reversed by expression of both yqgE+ and yqgF+.
at intragenic sites [2]. The knock-out of yqgF showed inviability of the organism suggesting that
yqgF is essential to E. coli [1].

In reverse transcriptase-PCR experiments, yqgE and yqgF were shown to be co-transcribed as

part of an operon. The yqgE25::IS1 insertion disrupts transcription of yqgEF, and cells grow
poorly at 37˚C and exhibit cold-sensitivity below 27˚C.

In this work, plasmid expressed yqgE+, yqgF+, or yqgE+F+ was used to attempt phenotype
restoration. The yqgE+F+ clone completely reversed the cold-sensitivity phenotype, suggesting
that the two gene products work together. Each gene was cloned into a different His6 expression
vector and the corresponding proteins were purified using NTA-Ni2+ agarose columns.

Figure 3. DNA used for transformations was isolated from a hsdR hsdM+ strain, then introduce
competent yqgE25::IS1 cells. Transformants were purified on selective media at 37 C, then pla
Figure 1. Overview of SOS regulation. 22 C on L-agar containing either D-glucose or L-arabinose (0.2%). Complementation was scor
comparing colony size of wildtype and mutant transformants after 84 hours.