scholarly journals A relationship between the starting secondary structure of recombinant porcine growth hormone solubilised from inclusion bodies and the yield of native (monomeric) protein after in vitro refolding

FEBS Letters ◽  
1992 ◽  
Vol 305 (3) ◽  
pp. 177-180 ◽  
Author(s):  
N.K. Puri ◽  
M. Cardamone
Keyword(s):  
Growth Hormone ◽  
Secondary Structure ◽  
Inclusion Bodies ◽  
In Vitro Refolding

scholarly journals Refolding and recognition of mitochondrial malate dehydrogenase by Escherichia coli chaperonins cpn 60 (groEL) and cpn10 (groES)

1994 ◽  
Vol 302 (2) ◽  
pp. 405-410 ◽  
Author(s):  
J P Hutchinson ◽  
T S el-Thaher ◽  
A D Miller
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Malate Dehydrogenase ◽  
Binding Assay ◽  
Specific Binding ◽  
Guanidinium Chloride ◽  
Mitochondrial Malate Dehydrogenase ◽  
In Vitro Refolding ◽  
Cpn 60

In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). When the enzyme is initially denatured with 3 M guanidinium chloride, chaperonin-assisted refolding is 100% efficient. C.d. spectroscopy reveals that malate dehydrogenase is almost unfolded in 3 M guanidinium chloride, suggesting that a state with little or no residual secondary structure is the optimal ‘substrate’ for chaperonin-assisted refolding. Malate dehydrogenase denatured to more highly structured states proves to refold less efficiently with chaperonin assistance. The enzyme is shown not to aggregate under the refolding conditions, so that losses in refolding efficiency result from irreversible misfolding. Evidence is advanced to suggest that the chaperonins are unable to rescue irreversibly misfolded malate dehydrogenase. A novel use is made of 100 K Centricon concentrators to study the binding of [14C]acetyl-labelled malate dehydrogenase to groEL by an ultrafiltration binding assay. Analysis of the data by Scatchard plot shows that acetyl-malate dehydrogenase, which has previously been extensively unfolded with guanidinium chloride, binds to groEL at a specific binding site(s). At saturation, one acetyl-malate dehydrogenase homodimer (two polypeptides) is shown to bind to each groEL homooligomer with a binding constant of approx. 10 nM.

scholarly journals Chemical Assistance in Refolding of Bacterial Inclusion Bodies

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Mona Alibolandi ◽  
Hasan Mirzahoseini
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Industrial Processes ◽  
Small Scale ◽  
Heterologous Proteins ◽  
E Coli ◽  
Major Bottleneck ◽  
In Vitro Refolding

Escherichia coliis one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins inE. coli.In vitrorefolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

scholarly journals Solubilization of growth hormone and other recombinant proteins from Escherichia coli inclusion bodies by using a cationic surfactant

1992 ◽  
Vol 285 (3) ◽  
pp. 871-879 ◽  
Author(s):  
N K Puri ◽  
E Crivelli ◽  
M Cardamone ◽  
R Fiddes ◽  
J Bertolini ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Cationic Surfactant ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Interleukin 1 ◽  
Exchange Chromatography ◽  
Head Group ◽  
Guanidinium Chloride ◽  
Native Page

Recombinant pig growth hormone (rPGH) was solubilized from inclusion bodies by using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The solubilizing action of CTAC appeared to be dependent on the presence of a positively charged head group, as a non-charged variant was inactive. Relatively low concentrations of CTAC were required for rapid solubilization, and protein-bound CTAC was easily removed by ion-exchange chromatography. Compared with solubilization and recovery of rPGH from inclusion bodies with 7.5 M-urea and 6 M-guanidinium chloride, the relative efficiency of solubilization was lower with CTAC. However, superior refolding efficiency resulted in final yields of purified rPGH being in the order of CTAC greater than urea greater than or equal to guanidinium chloride. Detailed comparison of the different rPGH preparations as well as pituitary-derived growth hormone by h.p.l.c., native PAGE, c.d. spectral analysis and radioreceptor-binding assay showed that the CTAC-derived rPGH was essentially indistinguishable from the urea and guanidinium chloride preparations. The CTAC-derived rPGH was of greater biopotency than pituitary-derived growth hormone. The advantages of CTAC over urea and guanidinium chloride for increasing recovery of monomeric rPGH by minimizing aggregation during refolding in vitro were also found with recombinant sheep interleukin-I beta and a sheep insulin-like growth factor II fusion protein. In addition, the bioactivity of the CTAC-derived recombinant interleukin-1 beta was approximately ten-fold greater than that of an equivalent amount obtained from urea and guanidinium chloride preparations. It is concluded that CTAC represents, in general, an excellent additional approach or a superior alternative to urea and in particular guanidinium chloride for solubilization and recovery of bioactive recombinant proteins from inclusion bodies.

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