New cloning and expression vector derived from Escherichia coli plasmid pIGWZ12; a potential vector for a two-plasmid expression system.
We constructed pIGPZ, a new cloning and expression vector derived from Escherichia coli plasmid pIGWZ12::Kan. pIGPZ contains a kanamycin resistance marker, a multiple-cloning-site (MCS) region, and a promoter for constitutive expression of cloned genes. pIGPZ has the same high level of stability as the original plasmid, even in the absence of antibiotic selection. Furthermore, we show that pIGPZ is compatible with ColE1-based plasmids and a pSC101-like plasmid. All the characteristic elements of theta-replicating plasmids were found in the pIGPZ putative origin of replication. Finally, we demonstrate that pIGPZ can be used in a double-plasmid expression system by co-expressing UBP1 protease from pIGPZ with ubi-interferon alpha (IFNA13; GenBank Accession No. NM_006900.3) or ubi-human growth hormone (ubi-hGH; patent No. WO 2005/066208 A2) cloned in another plasmid. In this system, both ubi-interferon alpha and ubi-human growth hormone were deubiquitinated efficiently in E. coli cells.
Institute of Biotechnology and Antibiotics, Warsaw, Poland. zaleskip@iba.waw.pl
Piotr
P
Zaleski
Pawel
P
Wawrzyniak
Agnieszka
A
Sobolewska
Diana
D
Mikiewicz
Anna
A
Wojtowicz-Krawiec
Luiza
L
Chojnacka-Puchta
Marcin
M
Zielinski
Grazyna
G
Plucienniczak
Andrzej
A
Plucienniczak
D003001:Cloning, Molecular; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007612:Kanamycin; D010957:Plasmids / Q000235:genetics*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D014157:Transcription Factors; C085968:UBP1 protein, human; D007612:Kanamycin