Purification and Characterization of Human and Mouse Recombinant Alpha-Fetoproteins Expressed inEscherichia coli

Boismenu, Richard; Semeniuk, Daniel; Murgita, Robert A.

doi:10.1006/prep.1996.0697

Cited by 27 publications

(27 citation statements)

References 40 publications

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“…This result is expected to resolve controversies on AFP foldability reported in the literature; a study on AFP molecular behavior during denaturation and refolding discusses the ''irreversibility'' of AFP denaturation (Gillespie and Uversky, 2000) and appears to contradict a separate earlier study which claimed to have successfully refolded the molecule from the denatured inclusion body state (Boismenu et al, 1997).…”

Section: Introductionmentioning

confidence: 76%

“…In contrast, chemical extraction achieves no initial separation of product from protease, although some proteases will be denatured during extraction. rhAFP is known to be sensitive to proteases (Boismenu et al, 1997), and it has also been reported that some proteins can be rapidly degraded by E. coli proteases active in denaturing conditions (Babbitt et al, 1991;Wong et al, 1996). While the E. coli strain BL21(DE3)RIL used in this work is deficient in the OmpT and lon proteases, the proteases responsible for degradation during the solubilization of impure preparations of inclusion bodies have not been precisely identified.…”

Section: Results and Discussion Rhafp Stability Against Proteolysismentioning

confidence: 99%

“…Boismenu et al (1997) reported that dilution refolding increased the recovery of soluble rhAFP, compared with dialysis refolding, although no direct quantitation of refolding yields was reported. Results from a separate dilution refolding study of std-rhAFP, showed that high refolding yield (>70%) could be easily achieved at a refolding protein concentration of 30 mg/mL (Leong and Middelberg, 2006), thus supporting the use of dilution refolding in this E. coli process.…”

Section: Design Choices For the Rhafp Processmentioning

confidence: 99%

“…Transgenically-derived rhAFP (from goats) is currently in clinical development by Merrimack Pharmaceuticals (Cambridge, MA), for the treatment of several autoimmune diseases. rhAFP manufacture using Escherichia coli (E. coli), has the potential to deliver a product having very low cost-of-goods, especially if the embedded complexity in the existing laboratory process (Boismenu et al, 1997) can be eliminated by successful chemical extraction coupled with appropriate contaminant removal methods. rhAFP is an ideal protein with which to address the contaminant removal challenge, as it binds contaminants easily once in its native state (Hsia et al, 1980;Parmelee et al, 1978).…”

Section: Introductionmentioning

confidence: 99%

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A simplified bioprocess for human alpha‐fetoprotein production from inclusion bodies

Leong

Middelberg

2006

Biotech & Bioengineering

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A simple and effective Escherichia coli (E. coli) bioprocess is demonstrated for the preparation of recombinant human alpha-fetoprotein (rhAFP), a pharmaceutically promising protein that has important immunomodulatory functions. The new rhAFP process employs only unit operations that are easy to scale and validate, and reduces the complexity embedded in existing inclusion body processing methods. A key requirement in the establishment of this process was the attainment of high purity rhAFP prior to protein refolding because (i) rhAFP binds easily to hydrophobic contaminants once refolded, and (ii) rhAFP aggregates during renaturation, in a contaminant- dependent way. In this work, direct protein extraction from cell suspension was coupled with a DNA precipitation-centrifugation step prior to purification using two simple chromatographic steps. Refolding was conducted using a single-step, redox-optimized dilution refolding protocol, with refolding success determined by reversed phase HPLC analysis, ELISA, and circular dichroism spectroscopy. Quantitation of DNA and protein contaminant loads after each unit operation showed that contaminant levels were reduced to levels comparable to traditional flowsheets. Protein microchemical modification due to carbamylation in this urea-based process was identified and minimized, yielding a final refolded and purified product that was significantly purified from carbamylated variants. Importantly, this work conclusively demonstrates, for the first time, that a chemical extraction process can substitute the more complex traditional inclusion body processing flowsheet, without compromising product purity and yield. This highly intensified and simplified process is expected to be of general utility for the preparation of other therapeutic candidates expressed as inclusion bodies.

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Section: Introductionmentioning

confidence: 76%

Section: Results and Discussion Rhafp Stability Against Proteolysismentioning

confidence: 99%

Section: Design Choices For the Rhafp Processmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A simplified bioprocess for human alpha‐fetoprotein production from inclusion bodies

Leong

Middelberg

2006

Biotech & Bioengineering

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“…A series of experiments showed that intraepithelial Á‰ T cells produce growth factors such as keratinocyte growth factor that may promote healing of damaged tissues [14]. Other studies of intestinal Á‰ T cells using dextran sulphate sodium-induced colitis, have now clearly established the importance of these cells in maintaining tissue homeostasis [15][16][17]. Moreover, the demonstration that intraepithelial Á‰ T cells produce a wide variety of chemokines and pro-inflammatory cytokines raises the possibility that Á‰ T cells also function to control immune reactions within mucosal tissues.…”

Section: á‰ T Cellsmentioning

confidence: 99%

Immunology

et al. 2004

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It has been known for the past 85 years that mucosal responses can be stimulated by local presentation of antigen and that the gut immune system is capable of mounting both primary and secondary responses to potentially harmful antigens while avoiding the expression of damaging responses to harmless dietary proteins. How these types of responses are induced and regulated remains unclear. In the gut attention has for some time been focused on Peyer’s patches (PP) due to evidence that they play a vital role in the induction of humoral and cellular responses. Moreover, it has been established that MHC class II molecules are found in the gut mucosa on a variety of cell types outside PP, namely the lamina propria (LP). Fed antigens have also been detected in the LP and studies have shown that LP cells can stimulate allogeneic mixed lymphocyte responses and are capable of presenting soluble protein antigen to naïve T cells. This article reviews the present understanding of the possible roles of PP and LP in intestinal immunity in terms of induction, regulation, surveillance of immune responses and the antigen presenting cell types involved.

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Immunology

et al. 2004

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It has been known for the past 85 years that mucosal responses can be stimulated by local presentation of antigen and that the gut immune system is capable of mounting both primary and secondary responses to potentially harmful antigens while avoiding the expression of damaging responses to harmless dietary proteins. How these types of responses are induced and regulated remains unclear. In the gut attention has for some time been focused on Peyer's patches (PP) due to evidence that they play a vital role in the induction of humoral and cellular responses. Moreover, it has been established that MHC class II molecules are found in the gut mucosa on a variety of cell types outside PP, namely the lamina propria (LP). Fed antigens have also been detected in the LP and studies have shown that LP cells can stimulate allogeneic mixed lymphocyte responses and are capable of presenting soluble protein antigen to naïve T cells. This article reviews the present understanding of the possible roles of PP and LP in intestinal immunity in terms of induction, regulation, surveillance of immune responses and the antigen presenting cell types involved.

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Purification and Characterization of Human and Mouse Recombinant Alpha-Fetoproteins Expressed inEscherichia coli

Cited by 27 publications

References 40 publications

A simplified bioprocess for human alpha‐fetoprotein production from inclusion bodies

A simplified bioprocess for human alpha‐fetoprotein production from inclusion bodies

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