Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease

Fu, Zhibiao; Hamid, Suhali Bt Ab; Razak, Che Nyonya Abdul; Basri, Mahiran; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Abd.

doi:10.1016/s1046-5928(02)00637-x

Cited by 43 publications

(30 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…According to Fu et al (2003) the enzyme activity was calculated as the total activity of 500 mL (11,000 U) that equal to 22 U/mL activities (result not shown). Nevertheless, the actual U/mL was 2.2 U/mL by comparing to the one unit definition used in current study (0.1/min/ mL).…”

Section: Transformation Into P Pastoris Strain Gs115 and Smd1168hmentioning

confidence: 99%

“…It can be isolated from various microbial sources (Bajaj and Sharma, 2011;Fu et al, 2003;Gohel and Singh, 2012;Purohit and Singh, 2011). Among all types of proteases, alkaline protease is the largest subgroup of serine protease enzymes and highly active at extreme alkaline pH.…”

Section: Introductionmentioning

confidence: 99%

“…Previously, the thermostable F1 protease was successfully cloned and expressed in Escherichia coli expression system extracellularly. A simple heattreatment method was used to purify the expressed protein (Fu et al, 2003). The lack of secretory pathway in E. coli system has encouraged the researchers to use of Edited by Rodney Rothstein * Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris

et al. 2013

Self Cite

View full text Add to dashboard Cite

The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme's capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa.

show abstract

Section: Transformation Into P Pastoris Strain Gs115 and Smd1168hmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris

et al. 2013

Self Cite

View full text Add to dashboard Cite

show abstract

“…BRP is a small lipoprotein of 28 amino acids, when expressed at high-level, it leads to quasi-lysis and lethality (Rahman et al 2005). Several proteins, such as b-lactamase, human immunoglobulin, penicillinase, penicillin acylase, F1 alkaline protease, and lipase, were secreted with the co-expression of BRP (Kitai et al 1988;Lurink et al 1987;Jin et al 2001;Fu et al 2003;Rahman et al 2005). Under optimized condition, at a rather low IPTG concentration (0.05 mM), presumably to reduce the effect of BRP on cell viability, the secretion of 18,100 U/ml thermostable T1 lipase was reported, demonstrating the effectiveness of the secretion through this method (Rahman et al 2005).…”

Section: Using Cell Envelope Mutantsmentioning

confidence: 99%

Extracellular recombinant protein production from Escherichia coli

Chen

2009

Biotechnol Lett

124

View full text Add to dashboard Cite

Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.

show abstract

“…Therefore, the most thoroughly characterized export systems of E. coli [19] are utilized for the expression of industrially important enzymes such as alkaline proteases. The recombinants of E. coli harboring the protease gene from Erwinia chrysanthemi and from Pseudomonas aeurginosa [20] have been shown to secrete recombinant proteins.…”

Section: Discussionmentioning

confidence: 99%

Partial Characterization and Cloning of Protease From Bacillus

Ramakrishnan

Thambidurai²,

Rajasekharan

et al. 2017

Asian J Pharm Clin Res

View full text Add to dashboard Cite

Objective: The present research focused on amplification of protease gene from Bacillus strain which was then assessed for maximal enzyme activity.Methods: A putative Bacillus strain was isolated from soil, inoculated into protease production media, and optimized with appropriate pH and temperature conditions for maximal enzyme activity. Genomic DNA was isolated from the strain and amplified the fragment by polymerase chain reaction (PCR) using gene-specific primers for protease. The fragment is then ligated into a T/A cloning vector and transformed into calcium chloridetreated competent Escherichia coli DH5α cells. The plasmids were then isolated and confirmed the presence of the gene.Results: A specific amplification of 1.1 kb was observed following PCR. The amplified product includes the coding sequence and a signal peptide sequence of the protease gene. After cloning with T/A cloning vector pTZ57R/T and transformed into E. coli DH5α competent cells, the recombinant plasmid was selected using blue-white selection. Plasmid DNA isolated from the recombinant strains and confirmed the presence of a gene of interest using PCR and quantified by an assay for maximal protease activity. The optimum pH was found to be 10.1 and giving an activity of 21.566 international unit (IU)/ml, and the optimum temperature was found to be on 60°C giving an activity of 38.708 IU/ml. Conclusion:Amplification of protease gene by PCR isolated from Bacillus strain and optimization of pH and temperature conditions for the assessment of subtilisin Carlsberg produced by it. Subtilisin which is protein engineered can be used in commercial products such as stain cutter, dishwashing detergents, cosmetics and food processing, and contact lens cleaner.

show abstract

Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease

Cited by 43 publications

References 12 publications

Secretory expression of thermostable alkaline protease from <i>Bacillus stearothermophilus</i> FI by using native signal peptide and <b>α</b>-factor secretion signal in <i>Pichia pastoris</i>

Secretory expression of thermostable alkaline protease from <i>Bacillus stearothermophilus</i> FI by using native signal peptide and <b>α</b>-factor secretion signal in <i>Pichia pastoris</i>

Extracellular recombinant protein production from Escherichia coli

Partial Characterization and Cloning of Protease From Bacillus

Contact Info

Product

Resources

About