Not Limited to E. coli: Versatile Expression Vectors for Mammalian Protein Expression

Author(s):  
Katharina Karste ◽  
Maren Bleckmann ◽  
Joop van den Heuvel
Author(s):  
N. Hemamalini ◽  
S. Ezhilmathi ◽  
A. Angela Mercy

Escherichia coli is the most extensively used organism in recombinant protein production. It has several advantages including a very short life cycle, ease of genetic manipulation and the well-known cell biology etc. which makes E. coli as the perfect host for recombinant protein expression. Despite many advantages, E. coli also have few disadvantages such as coupled transcription and translation and lack of eukaryotic post-translational modifications. These challenges can be overcome by adopting several strategies such as, using different E. coli expression vectors, changing the gene sequence without altering the functional domain, modified E. coli strain usage, changing the culture parameters and co-expression with a molecular chaperone. In this review, we present the level of strategies used to enhance the recombinant protein expression and its stability in E. coli.


2010 ◽  
Vol 5 (1) ◽  
pp. 11 ◽  
Author(s):  
Alimuddin Alimuddin ◽  
Indra Lesmana ◽  
Agus Oman Sudrajat ◽  
Odang Carman ◽  
Irvan Faizal

This study was aimed to produce recombinant growth hormone (rGH) from giant grouper (Epinephelus lanceolatus), giant gouramy (Osphronemus gouramy) and common carp (Cyprinus carpio) and compare their bioactivity potential by means of inducing the growth hormone of juvenile Nile tilapia (Oreochromis niloticus) as the model. DNA fragment encoding mature GH protein of giant grouper (El-mGH), giant gouramy (Og-mGH) and common carp (Cc-mGH) was amplified by PCR method. The purified PCR products were ligated to pCold-1 to generate pCold/El-mGH, pCold/OgmGH, and pCold/Cc-mGH protein expression vector, respectively. Each of the expression vectors was transformed into the Escherichia coli BL21. E. coli BL21 was cultured using 2xYT medium and protein production was induced by cold shock at 15±1oC for overnight. The inclusion bodies of E. coli transformants containing protein expression vector were isolated by sonication method, and rGH production was analyzed by SDS-PAGE. Juvenile of Nile tilapia of average body weight of 12.41±3.28 g was intramuscularly injected once a week for 4 weeks with 1 μg inclusion body containing rGH per gram fish body weight. The result showed that rGH in molecular weight of about 25 kDa was obtained. Fish injected with rGH of El-mGH, Cc-mGH and Og-mGH grew 20.94%, 18.09%, and 16.99% faster, respectively, compared with the control. This result indicated that the three rGH produced in E. coli possessed biological activity when tested on Nile tilapia and further research is needed to find its effect on the growth of other aquaculture fish species.


2003 ◽  
pp. 19-30
Author(s):  
Rebecca L. Mullinax ◽  
David T. Wong ◽  
Heidi A. Davis ◽  
Kerstein A. Padgett ◽  
Joseph A. Sorge

2021 ◽  
Vol 22 (12) ◽  
pp. 6415
Author(s):  
Barbara Jana ◽  
Jarosław Całka ◽  
Bartosz Miciński

Uterine inflammation is a very common and serious pathology in domestic animals, the development and progression of which often result from disturbed myometrial contractility. We investigated the effect of inflammation on the protein expression of galanin (GAL) receptor subtypes (GALR)1 and GALR2 in myometrium and their role in the contractile amplitude and frequency of an inflamed gilt uterus. The gilts of the E. coli and SAL groups received E. coli suspension or saline in their uteri, respectively, and only laparotomy was performed (CON group). Eight days later, the E. coli group developed severe acute endometritis and lowered GALR1 protein expression in the myometrium. Compared to the pretreatment period, GAL (10−7 M) reduced the amplitude and frequency in myometrium and endometrium/myometrium of the CON and SAL groups, the amplitude in both stripes and frequency in endometrium/myometrium of the E. coli group. In this group, myometrial frequency after using GAL increased, and it was higher than in other groups. GALR2 antagonist diminished the decrease in amplitude in myometrium and the frequency in endometrium/myometrium (SAL, E. coli groups) induced by GAL (10−7 M). GALR1/GALR2 antagonist and GAL (10−7 M) reversed the decrease in amplitude and diminished the decrease in frequency in both examined stripes (CON, SAL groups), and diminished the drop in amplitude and abolished the rise in the frequency in the myometrium (E. coli group). In summary, the inflammation reduced GALR1 protein expression in pig myometrium, and GALR1 and GALR2 participated in the contractile regulation of an inflamed uterus.


1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.


Mycologia ◽  
2005 ◽  
Vol 97 (5) ◽  
pp. 1152-1161 ◽  
Author(s):  
R. M. Andrie ◽  
J. P. Martinez ◽  
L. M. Ciuffetti

2005 ◽  
Vol 70 (12) ◽  
pp. 1401-1407 ◽  
Author(s):  
Sandra Markovic ◽  
Sandra Vojnovic ◽  
Milija Jovanovic ◽  
Branka Vasiljevic

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.


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