A novel Method for the selective recovery and purification of γ‐polyglutamic acid from Bacillus licheniformis fermentation broth

Microbially produced gamma‐polyglutamic acid (γ‐PGA) is a commercially important biopolymer with many applications in biopharmaceutical, food, cosmetic and waste‐water treatment industries. Owing to its increasing demand in various industries, production of γ‐PGA is well documented in the literature, however very few methods have been reported for its recovery. In this paper, we report a novel method for the selective recovery and purification of γ‐PGA from cell‐free fermentation broth of Bacillus licheniformis. The cell‐free fermentation broth was treated with divalent copper ions, resulting in the precipitation of γ‐PGA, which was collected as a pellet by centrifugation. The pellet was resolubilized and dialyzed against de‐ionized water to obtain the purified γ‐PGA biopolymer. The efficiency and selectivity of γ‐PGA recovery was compared with ethanol precipitation method. We found that 85% of the original γ‐PGA content in the broth was recovered by copper sulfate‐induced precipitation, compared to 82% recovery by ethanol precipitation method. Since ethanol is a commonly used solvent for protein precipitation, the purity of γ‐PGA precipitate was analyzed by measuring proteins that co‐precipitated with γ‐PGA. Of the total proteins present in the broth, 48% proteins were found to be co‐precipitated with γ‐PGA by ethanol precipitation, whereas in copper sulfate‐induced precipitation, only 3% of proteins were detected in the final purified γ‐PGA, suggesting that copper sulfate‐induced precipitation offers better selectivity than ethanol precipitation method. Total metal content analysis of the purified γ‐PGA revealed the undetectable amount of copper ions, whereas other metal ions detected were in low concentration range. The purified γ‐PGA was characterized using infrared spectroscopy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effects of solution environment on mammalian cell fermentation broth properties: Enhanced impurity removal and clarification performance

The processing of recombinant proteins from high cell density, high product titer cell cultures containing mammalian cells is commonly performed using tangential flow microfiltration (MF). However, the increased cellular debris present in these complex feed streams can prematurely foul the membrane, adversely impacting MF capacity and throughput. In addition, high cell density cell culture streams introduce elevated levels of process‐related impurities, which increase the burden on subsequent purification operations to remove these complex media components and impurities. To address this challenge, an evaluation of mammalian cell culture broth buffer properties was examined to determine if enhanced impurity removal and clarification performance could be achieved. A framework is presented here for establishing optimized mammalian cell culture buffer conditions, involving trade‐offs between product recovery and purification and improved clarification at manufacturing‐scale production. A reduction in cell culture broth pH to 4.7–5.0 induced flocculation and impurity precipitation which increased the average feed particle‐size. These conditions led to enhanced impurity removal and improved MF throughput and filter capacity for several mammalian systems. Feed conditions were further optimized by controlling ionic composition along with pH to improve product recovery from high cell density/high product titer cell cultures. Biotechnol. Bioeng. 2011; 108:50–58. © 2010 Wiley Periodicals, Inc.     

Control of misincorporation of serine for asparagine during antibody production using CHO cells

A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed‐batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time‐course analysis of cell culture suggest that misincorporation was occurring midway through the fed‐batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench‐scale culture via controlled supplementation of asparagine‐containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales. Biotechnol. Bioeng. 2010;107: 116–123. © 2010 Wiley Periodicals, Inc.     

l-Arginine hydrochloride increases the solubility of folded and unfolded recombinant plasminogen activator rPA

L ‐Arginine hydrochloride (L ‐ArgHCl) was found to be an effective enhancer for in vitro protein refolding more than two decades ago. A detailed understanding of the mechanism of action, by which L ‐ArgHCl as co‐solvent is capable to effectively suppress protein aggregation, while protein stability is preserved, has remained elusive. Concepts for the effects of co‐solvents, which have been established over the last decades, were found to be insufficient to completely explain the effects of L ‐ArgHCl on protein refolding. In this article, we present data, which clearly establish that L ‐ArgHCl acts on the equilibrium solubility of the native model protein recombinant plasminogen activator (rPA), while for S‐carboxymethylated rPA (IAA‐rPA) that served as a model protein for denatured protein states, equilibrium solubilities could not be obtained. Solid to solute free transfer energies for native rPA were lowered by up to 14 kJ mol^‐1 under the tested conditions. This finding is in marked contrast to a previously proposed model in which L ‐ArgHCl acts as a neutral crowder which exclusively has an influence on the stability of the transition state of aggregation. The effects on the apparent solubility of IAA‐rPA, as well as on the aggregation kinetics of all studied protein species, that were observed in the present work could tentatively be explained within the framework of a nucleation‐aggregation scheme, in which L ‐ArgHCl exerts a strong effect on the pre‐equilibria leading to formation of the aggregation seed.     

Continuous capture of recombinant antibodies by ZnCl2 precipitation without polyethylene glycol

The capture of recombinant antibodies from cell culture broth is the first critical step of downstream processing. We were able to develop a precipitation‐based method for the capture and purification of monoclonal antibodies based on divalent cations, namely ZnCl₂. Traditional precipitation processes have to deal with high dilution factors especially for resolubilization and higher viscosity due to the use of PEG as precipitation or co‐precipitation agent. By the use of the crosslinking nature of divalent cations without the use of PEG, we kept viscosity from the supernatant and resolubilization dilution factors very low. This is especially beneficial for the solid–liquid separation for the harvest and wash of the precipitate in continuous mode. For this harvest and wash, we used tangential flow filtration that benefits a lot from low viscosity solutions, which minimizes the membrane fouling. With this precipitation based on ZnCl_2, we were able to implement a very lean and efficient process. We demonstrated precipitation studies with three different antibodies, Adalimumab, Trastuzumab, and Denosumab, and a continuous capture case study using tangential flow filtration for precipitate recovery. In this study, we achieved yields of 70%.     

Comparative kinetics of fatty acid-amino acid conjugate elicitor biosynthesis by midgut tissue microsomes of Lepidopterous caterpillar larvae

N‐Linolenoyl‐L ‐glutamine is one of several structurally similar fatty acid–amino acid conjugate (FAC) elicitors found in the oral secretions of Lepidopterous caterpillars and its biosynthesis is catalyzed by membrane‐associated alimentary tissue enzyme(s). FAC elicitors comprise 17‐hydroxylated or non‐hydroxylated linolenic acid coupled with L ‐glutamine or L ‐glutamate by an amide bond. We demonstrate in vitro biosynthesis of N‐linolenoyl‐L ‐glutamine by Manduca sexta, Heliothis virescens, and Helicoverpa zea tissue microsomes. Comparison of N‐linolenoyl‐L ‐glutamine biosynthesis kinetics for these species suggests that concurrent biosynthesis and hydrolysis contribute to proportions of FAC elicitors found in their oral secretions. The apparent K_m values for coupling of sodium linolenate were 8.75±0.79, 14.3±3.7 and 20.7±3.4 mM and V_max values were 2.92±0.14, 6.81±1.2 and 4.95±0.55 nmol/min/mg protein for H. zea, H. virescens and M. sexta, respectively. The K_m values for coupling of L ‐glutamine were 10.5±0.26, 22.3±2.0 and 18.9±2.4 mM and V_max values were 1.78±0.21, 3.71±0.50 and 2.49±0.41 nmol/min/mg of protein for H. zea, H. virescens and M. sexta, respectively. © 2010 Wiley Periodicals, Inc.     

Cytokine production using membrane adsorbers: Human basic fibroblast growth factor produced by Escherichia coli

Basic fibroblast growth factor (FGF‐2) is a multifunctional cytokine that regulates various cellular processes both in vitro and in vivo. FGF‐2 is extensively used in embryonic stem cell cultures since it can maintain the cells in an undifferentiated state. However, the high price of FGF‐2 has limited its application in stem cell research. Here we present a fast and efficient process for the purification of FGF‐2 from recombinant Escherichia coli cultures using reusable membrane adsorbers. A high expression level of FGF‐2 (42 mg/g dry cell) was achieved by fed‐batch cultivation of E. coli BL21(DE3). A new combination of cation exchange membrane chromatography and heparin‐sepharose affinity chromatography was used for the purification of the protein. A novel anion exchange membrane chromatography was used in the polishing step to remove endotoxins and DNA. In this new process, about 200 mg soluble FGF‐2 was yielded from 1.9 L culture broth with a purity of 98%. The purified protein was identified to be endotoxin‐free and bioactive. It was successfully tested to keep primate embryonic stem cell and human‐induced pluripotent stem cell pluripotent. Our approach, in which a controlled cultivation process is combined with an optimized fast and versatile downstreaming process, is suitable for low‐cost preparation of bioactive FGF‐2 at bench‐scale and may be beneficial to the effective production of other cytokines.     

A study of D‐lactate and extracellular methylglyoxal production in lactate Re‐Utilizing CHO cultures

In large‐scale mammalian cell culture, the key toxic by‐products assessed and monitored are lactate and ammonia. Often no distinction between the two isoforms of lactate is made. Here, we present profiles of both D ‐ and L ‐lactate. D ‐Lactate is the end molecule of the methylglyoxal pathway. D ‐Lactate unlike L ‐lactate is not re‐utilized, and although during normal culture time frames it represents one‐tenth of total lactate, during lactate re‐use it represents nearly 35%. This indicates significant carbon flow through pathways not associated with primary metabolites. We have observed that the behavior of D ‐lactate is radically different from that of L ‐lactate with the level of one isoform changing, whilst the concentration of the other remains constant. This is an example of an alternate carbon flow pathway containing metabolic intermediates that may potentially have a detrimental effect on cells. The activity of the methylglyoxal pathway when measured as a proportion of glucose consumption in this study far exceeds any previously reported. This highlights the potential importance of “non‐primary” metabolisms to long lifespan mammalian fermentation practices. Biotechnol. Bioeng. 2010;107: 182–189. © 2010 Wiley Periodicals, Inc.     

Fed‐batch CHO cell t‐PA production and feed glutamine replacement to reduce ammonia production

Industrial therapeutic protein production has been greatly improved through fed‐batch development. In this study, improvement to the productivity of a tissue‐plasminogen activator (t‐PA) expressing Chinese hamster ovary (CHO) cell line was investigated in shake flask culture through the optimization of the fed‐batch feed and the reduction of ammonia generation by glutamine replacement. The t‐PA titer was increased from 33 mg/L under batch conditions to 250 mg/L with daily feeding starting after three days of culture. A commercially available fed‐batch feed was supplemented with cotton seed hydrolysate and the four depleted amino acids, aspartic acid, asparagine, cysteine, and tyrosine. The fed‐batch operation increased the generation of by‐products such as lactate and ammonia that can adversely affect the fed‐batch performance. To reduce the ammonia production, a glutamine‐containing dipeptide, pyruvate, glutamate, and wheat gluten hydrolysate, were investigated as glutamine substitutes. To minimize the lag phase as the cells adjusted to the new energy source, a feed glutamine replacement process was developed where the cells were initially cultured with a glutamine containing basal medium to establish cell growth followed by feeding with a feed containing the glutamine substitutes. This two‐step feed glutamine replacement process not only reduced the ammonia levels by over 45% but, in the case of using wheat gluten hydrolysate, almost doubled the t‐PA titer to over 420 mg/L without compromising the t‐PA product quality or glycosylation pattern. The feed glutamine replacement process combined with optimizing other feed medium components provided a simple, practical, and effective fed‐batch strategy that could be applied to the production of other recombinant therapeutic proteins. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Extracellular production of human parathyroid hormone as a thioredoxin fusion form in Escherichia coli by chemical permeabilization combined with heat treatment

A pET system encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH) was introduced into Escherichia coli BL21 (DE3). Recombinant Trx-hPTH fusion protein was expressed in soluble form in the cytoplasm of the E. coli transformant. To recover Trx-hPTH from the E. coli culture efficiently, a novel tactic was developed by adding Triton X-100 into the fermentation culture at the exponential growth phase of E. coli and by heat treatment of the culture at the end of the fermentation. A concentration of 1% (v/v) Triton X-100 was added into the culture at the same time as IPTG addition after optimization. Under these conditions, addition of Triton X-100 had little effect on the cell growth, but more than 75% of the total recombinant Trx-hPTH was released into the fermentation broth. Also, a much higher volumetric yield of recombinant Trx-hPTH could be obtained with protein release compared to yield without protein release. Simultaneously, owing to the highly thermal stability of Trx-hPTH fusion protein, heat treatment of the fermentation broth at 80 degrees C for 15 min at the end of fermentation was employed for primary purification. Results demonstrated that heat treatment not only boosted further release of the recombinant Trx-hPTH fusion protein into the fermentation broth but also precipitated/denatured most of the nontarget proteins released in the broth. The tactics described herein integrated the fermentation process with subsequent recovery steps and thus provided a valuable and economical method for the production of Trx-hPTH and maybe some other Trx fusions in E. coli.     

Antioxidant efficacy and adhesion rescue by a recombinant mussel foot protein‐6

Mytilus foot protein type 6 (mfp‐6) is crucial for maintaining the reducing conditions needed for optimal wet adhesion in marine mussels. In this report, we describe the expression and production of a recombinant Mytilus californianus foot protein type 6 variant 1 (rmfp‐6.1) fused with a hexahistidine affinity tag in Escherichia coli and its purification by affinity chromatography. Recombinant mfp‐6 showed high purification yields of 5–6 mg L^?1 cell culture and excellent solubility in low pH buffers that retard oxidation of its many thiol groups. Purified rmfp‐6.1 protein showed high 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging activity when compared with vitamin C. Using the highly sensitive surface forces apparatus (SFA) technique to measure interfacial surface forces in the nano‐Newton range, we show that rmfp‐6.1 is also able to rescue the oxidation‐dependent adhesion loss of mussel foot protein 3 (mfp‐3) at pH 3. The adhesion rescue is related to a reduction of dopaquinone back to 3,4‐dihydroxyphenyl‐l ‐alanine in mfp‐3, which is the reverse reaction observed during the detrimental enzymatic browning process in fruits and vegetables. Broadly viewed, rmfp‐6.1 has potential as a versatile antioxidant for applications ranging from personal products to antispoilants for perishable foods during processing and storage. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1587–1593, 2013     

Evaluation of substrate promiscuity of an L‐carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43

N‐carbamoyl‐amino‐acid amidohydrolase (also known as N‐carbamoylase) is the stereospecific enzyme responsible for the chirality of the D ‐ or L ‐amino acid obtained in the “Hydantoinase Process.” This process is based on the dynamic kinetic resolution of D ,L ‐5‐monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L ‐N‐carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N‐acetyl and N‐formyl‐L ‐amino acids as well as the known N‐carbamoyl‐L ‐amino acids, thus proving its substrate promiscuity. BsLcar showed faster hydrolysis for N‐formyl‐L ‐amino acids than for N‐carbamoyl and N‐acetyl‐L ‐derivatives, with a catalytic efficiency (k_cat/K_m) of 8.58 × 10⁵, 1.83 × 10⁴, and 1.78 × 10³ (s^?1 M^?1), respectively, for the three precursors of L ‐methionine. Optimum reaction conditions for BsLcar, using the three N‐substituted‐L ‐methionine substrates, were 65°C and pH 7.5. In all three cases, the metal ions Co²⁺, Mn²⁺, and Ni²⁺ greatly enhanced BsLcar activity, whereas metal‐chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co²⁺‐dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.     

Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods.     

Effects of supplementation with free glutamine and the dipeptide alanyl‐glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise

In this study, we investigated the effect of the supplementation with the dipeptide L ‐alanyl‐L ‐glutamine (DIP) and a solution containing L ‐glutamine and L ‐alanine on plasma levels markers of muscle damage and levels of pro‐inflammatory cytokines and glutamine metabolism in rats submitted to prolonged exercise. Rats were submitted to sessions of swim training for 6 weeks. Twenty‐one days prior to euthanasia, the animals were supplemented with DIP (n = 8) (1.5 g.kg^?1), a solution of free L ‐glutamine (1 g.kg^?1) and free L ‐alanine (0.61 g.kg^?1) (G&A, n = 8) or water (control (CON), n = 8). Animals were killed at rest before (R), after prolonged exercise (PE—2 h of exercise). Plasma concentrations of glutamine, glutamate, tumour necrosis factor‐α (TNF‐α), prostaglandin E2 (PGE2) and activity of creatine kinase (CK), lactate dehydrogenase (LDH) and muscle concentrations of glutamine and glutamate were measured. The concentrations of plasma TNF‐α, PGE2 and the activity of CK were lower in the G&A‐R and DIP‐R groups, compared to the CON‐R. Glutamine in plasma (p < 0.04) and soleus muscle (p < 0.001) was higher in the DIP‐R and G&A‐R groups relative to the CON‐R group. G&A‐PE and DIP‐PE groups exhibited lower concentrations of plasma PGE2 (p < 0.05) and TNF‐α (p < 0.05), and higher concentrations of glutamine and glutamate in soleus (p < 0.001) and gastrocnemius muscles (p < 0.05) relative to the CON‐PE group. We concluded that supplementation with free L ‐glutamine and the dipeptide LL ‐alanyl‐LL ‐glutamine represents an effective source of glutamine, which may attenuate inflammation biomarkers after periods of training and plasma levels of CK and the inflammatory response induced by prolonged exercise. Copyright © 2009 John Wiley & Sons, Ltd.     

Fed-batch culture of Escherichia coli for L-valine production based on in silico flux response analysis

We have previously reported the development of a 100% genetically defined engineered Escherichia coli strain capable of producing L ‐valine from glucose with a high yield of 0.38 g L ‐valine per gram glucose (0.58 mol L ‐valine per mol glucose) by batch culture. Here we report a systems biological strategy of employing flux response analysis in bioprocess development using L ‐valine production by fed‐batch culture as an example. Through the systems‐level analysis, the source of ATP was found to be important for efficient L ‐valine production. There existed a trade‐off between L ‐valine production and biomass formation, which was optimized for the most efficient L ‐valine production. Furthermore, acetic acid feeding strategy was optimized based on flux response analysis. The final fed‐batch cultivation strategy allowed production of 32.3 g/L L ‐valine, the highest concentration reported for E. coli. This approach of employing systems‐level analysis of metabolic fluxes in developing fed‐batch cultivation strategy would also be applicable in developing strategies for the efficient production of other bioproducts. Biotechnol. Bioeng. 2011; 108:934–946. © 2010 Wiley Periodicals, Inc.     

Purification of recombinant protein by cold‐coacervation of fusion constructs incorporating resilin‐inspired polypeptides

Polypeptides containing between 4 and 32 repeats of a resilin‐inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence‐related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold‐coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein‐poor phase. We show that purification of recombinant proteins by cold‐coacervation can be performed when engineered as a fusion partner to a resilin‐inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time‐consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold‐coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. Biotechnol. Bioeng. 2012; 109: 2947–2954. © 2012 Wiley Periodicals, Inc.     

A pH‐induced,intein‐mediated expression and purification of recombinant human epidermal growth factor in Escherichia coli

Human epidermal growth factor (hEGF) is a cellular factor that promotes cell proliferation and has been widely used for the treatment of wounds, corneal injuries, and gastric ulcers. Recombinant hEGF (rhEGF) has previously been expressed using the pTWIN1 system with pH‐induced intein and a chitin‐binding domain. The rhEGF protein can be purified by chitin affinity chromatography because of the high affinity between the chitin‐binding domain fusion‐tag and the column. However, uncontrolled cleavage presents a major problem with this method. To overcome this problem, a novel purification method has been developed for a pH‐induced intein tag rhEGF that is expressed in Escherichia coli. Following purification by denaturation of inclusion bodies, the fusion protein is renatured and simultaneously induced to self‐cleave by dialysis. Further purification of rhEGF is achieved by heat treatment and ion‐exchange chromatography. Our results show that the purity of rhEGF obtained through this method is over 98% and the quantity of purified rhEGF is 248 mg from a 1 L culture or 2,967 mg from a 12 L culture. Therefore, we conclude that we have developed an efficient purification method of rhEGF, which may be used for the purification of other heat‐resistant and acid‐resistant recombinant proteins. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:758–764, 2015