High expression of cloned genes in E. coli and its consequences

Carrier, Martin; Nugent, Marilyn E.; Tacon, William C.; Primrose, Sandy B.

doi:10.1016/0167-7799(83)90033-1

Cited by 44 publications

(15 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The expressions of genes cloned downstream of the lac promoter can be conveniently induced by the addition of IPTG (de Bellis and Schwartz, 1990). All of the host cells used had the lac I q genotype to limit the background expression of the lac promoter, to ensure that Fab expression only occurs upon induction by IPTG, as constitutive expression of Fab C37 is likely to be detrimental and debilitating to the host (Carrier et al, 1993). Fab C37 expression was optimal after incubating for 6 h at 37 o C in the presence of a final concentration of 2 mM IPTG.…”

Section: Expression Purification and Characterization Of Fab C37mentioning

confidence: 99%

See 1 more Smart Citation

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Chan¹,

Ong²,

Nathan³

2004

BMB Reports

View full text Add to dashboard Cite

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

show abstract

Section: Expression Purification and Characterization Of Fab C37mentioning

confidence: 99%

“…Nevertheless, the overproduction of foreign proteins encoded by the DNA introduced into the host cell triggers the metabolic burden phenomenon (Carrier et al, 1993). Metabolite overload occurs when host cell ATP or GTP and certain amino acids are used to stabilize and express recombinant protein.…”

Section: Expression Purification and Characterization Of Fab C37mentioning

confidence: 99%

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Chan¹,

Ong²,

Nathan³

2004

BMB Reports

View full text Add to dashboard Cite

show abstract

“…Its biosynthesis is repressed by high concentrations of inorganic phosphate and can be regulated to start only towards the end of the logarithmic growth phase, simply by choosing the proper initial phosphate concentration 1271. This kind of regulation has been shown to be important in cases where the protein product of a recombinant DNA is detrimental to the host cells [28].…”

Section: Recombinant Plasmids Coding For Phosphatase-adrenocorticotromentioning

confidence: 99%

Production of human adrenocorticotropin by cleavage of alkaline‐phosphatase‐derived fusion proteins containing repetitive recognition sequences for collagenases

Daum¹,

Donner²,

Geilen³

et al. 1989

European Journal of Biochemistry

View full text Add to dashboard Cite

Recombinant plasmids coding for fusion proteins which consist of human adrenocorticotropin joined to Nterminal sequences of Escherichia coli alkaline phosphatase via collgenase-sensitive linkers were constructed and used for the production of these proteins by transformed E. coli cells. It was shown that repetitive linkers of the form -Gly-(Pro-Xaa-Gly),-Pro-with n 2 were cleaved by clostridiopeptidase A (Clostridium histolyticum) by orders of magnitude faster than corresponding nonrepetitive sequences (n = 1). The C-terminal cleavage product was Gly-Pro-adrenocorticotropin which could be converted to the authentic hormone by dipeptidyl peptidase IV. On the basis of these enzymatic reactions a procedure for the preparation of pure adrenocorticotropin was developed. Derivatives of alkaline phosphatase containing similar repetitive linker sequences were cleaved by clostridiopeptidase A as efficiently as the adrenocorticotropin fusion proteins.

show abstract

“…This type of construction provides an optimal sequence for translation initiation. The efficiency of translation is also affected by many other factors such as the choice of promoter, the sequence of bases in the vicinity of the ribosome binding site, the plasmid copy number, the codon usage, the presence of a transcription terminator, the choice of bacterial host, and the growth conditions of the bacterial host (Iserentant & Fiers, 1980;Shepard et al, 1982;Carrier et al, 1983;DeBoer & Hui, 1990;Goeddel, 1990).…”

mentioning

confidence: 99%

Increased production of low molecular weight recombinant proteins in Escherichia coli

Belagaje

Reams

et al. 1997

Protein Science

View full text Add to dashboard Cite

A general method for obtaining high-level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met-X,,-protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N-terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high-level production of human insulin-like growth factor-1, human proinsulin, and their analogs. Furthermore, highly produced insulin-like growth factor-] derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.

show abstract

High expression of cloned genes in E. coli and its consequences

Cited by 44 publications

References 15 publications

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Production of human adrenocorticotropin by cleavage of alkaline‐phosphatase‐derived fusion proteins containing repetitive recognition sequences for collagenases

Increased production of low molecular weight recombinant proteins in Escherichia coli

Contact Info

Product

Resources

About