The kinetics of RCC1 inclusion body formation in <i>Escherichia Coli</i>

Tsai, Amos M.; Betenbaugh, Michael J; Shiloach, Joseph

doi:10.1002/bit.260480620

Cited by 7 publications

(2 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Maintaining temperature below growth optimum has been used to decrease the rate of recombinant protein production, which in some cases results in larger proportions of proteins in a soluble form (Schein and Noteborn, 1988;Strandberg and Enfors, 1991;Tsai et al, 1995;Cicek and Esen, 1999). In three experiments (results not shown), IPTG was added to the GST-PPAR␣ LBD construct with a simultaneous decrease in temperature from 30 to 25°C.…”

Section: Discussionmentioning

confidence: 98%

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on‐line estimation of biomass concentration and specific growth rate

Eriksen

Kratchmarova

Neve

et al. 2001

Biotech & Bioengineering

View full text Add to dashboard Cite

This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli. Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH. When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically. The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S-transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPARalpha LBD. Total yield of GST-PPARalpha LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPARalpha LBD, the only active form, increased with the length of induction period. Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 +/- 0.4 cell divisions. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific growth rate was observed when inducer was added in the lag-phase, and no soluble protein was produced. These results suggest that solely soluble GST-PPARalpha LBD acts as a growth inhibitor and that GST-PPARalpha LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction. Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth.

show abstract

Section: Discussionmentioning

confidence: 98%

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on‐line estimation of biomass concentration and specific growth rate

Eriksen

Kratchmarova

Neve

et al. 2001

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…Many have found it advantageous to induce at low cell density in shaker flask culture to avoid growth limitation by the medium. The concentration of inducer may also be important; several authors have reported better yields of soluble protein if the isopropyl-D-thiogalactopyranoside (IPTG) inducer concentration was reduced from the usual 1 mM (44,45,93). Purify fusion protein by binding to amylose beads and eluting with maltose.…”

Section: Induction Conditionsmentioning

confidence: 99%

Soluble Protein Expression in Bacteria

Schein¹

2010

Encyclopedia of Industrial Biotechnology

View full text Add to dashboard Cite

This article summarizes methods for increasing the fraction of recombinant proteins expressed in a soluble form in the cytoplasmic fraction of bacteria. Many recombinant proteins, when expressed at very high levels in bacteria, form insoluble aggregates called inclusion bodies (IBs). Growth conditions can be manipulated to keep protein in a soluble form, including lowering the temperature during induction, choice of host strain and expression vector, or coculturing of proteins that assist in folding. Higher yields can be obtained by mutating the protein or fusing it with various linkers (which can also improve purification by enhancing column chromatography interactions). Eliminating protease or RNase recognition sites can stabilize the protein or mRNA, as can mutations that improve recognition by cofactors that assist in folding. Methodology to simultaneously produce many different recombinant proteins in a soluble form has been developed as part of the structural genomics initiative; these can also be used to for scanning mutagenesis studies. High density bacterial cultivation can be used to produce gram per liter quantities of recombinant proteins at the industrial scale. However, maintaining high levels of soluble protein requires optimizing all aspects of growth, ranging from how the inoculum is stored, to medium details and bacterial harvesting and lysis.

show abstract

Protein Expression, Soluble

Schein

1999

Encyclopedia of Bioprocess Technology

View full text Add to dashboard Cite

The kinetics of RCC1 inclusion body formation in Escherichia Coli

Cited by 7 publications

References 27 publications

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on‐line estimation of biomass concentration and specific growth rate

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on‐line estimation of biomass concentration and specific growth rate

Soluble Protein Expression in Bacteria

Protein Expression, Soluble

Contact Info

Product

Resources

About