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Purification, characterization and thermal inactivation kinetics of a non-regioselective thermostable lipase from a genotypically identified extremophilic Bacillus subtilis NS 8

Olusesan AT, Azura LK, Forghani B, Bakar FA, Mohamed AK, Radu S, et al.

N Biotechnol, 2011 Oct;28(6):738-45.
PMID: 21238617 DOI: 10.1016/j.nbt.2011.01.002

Thermostable lipase produced by a genotypically identified extremophilic Bacillus subtilis NS 8 was purified 500-fold to homogeneity with a recovery of 16% by ultrafiltration, DEAE-Toyopearl 650M and Sephadex G-75 column. The purified enzyme showed a prominent single band with a molecular weight of 45 kDa. The optimum pH and temperature for activity of lipase were 7.0 and 60°C, respectively. The enzyme was stable in the pH range between 7.0 and 9.0 and temperature range between 40 and 70°C. It showed high stability with half-lives of 273.38 min at 60°C, 51.04 min at 70°C and 41.58 min at 80°C. The D-values at 60, 70 and 80°C were 788.70, 169.59 and 138.15 min, respectively. The enzyme's enthalpy, entropy and Gibb's free energy were in the range of 70.07-70.40 kJ mol(-1), -83.58 to -77.32 kJ mol(-1)K(-1) and 95.60-98.96 kJ mol(-1), respectively. Lipase activity was slightly enhanced when treated with Mg(2+) but there was no significant enhancement or inhibition of the activity with Ca(2+). However, other metal ions markedly inhibited its activity. Of all the natural vegetable oils tested, it had slightly higher hydrolytic activity on soybean oil compared to other oils. On TLC plate, the enzyme showed non-regioselective activity for triolein hydrolysis.

Matched MeSH terms: Bacterial Proteins/isolation & purification*
Assessment of Clarias batrachus as a source of acetylcholinesterase (AchE) for the detection of insecticides

Tham LG, Perumal N, Syed MA, Shamaan NA, Shukor MY

J Environ Biol, 2009 Jan;30(1):135-8.
PMID: 20112875

An inhibitive assay of insecticides using Acetylcholinesterase (AChE) from the local fish Clarias batrachus is reported. AChE was assayed according to the modified method of Ellman. Screening of insecticide and heavy metals showed that carbofuran and carbaryl strongly inhibited C. batrachus AChE. The inhibition concentration (IC) IC50 values (and the 95% confidence interval) for both carbofuran and carbaryl inhibition on C. batrachus AChE at 6.66 (5.97-7.52) and 130.00 (119.3-142.5) microg l(-1), respectively was within the IC50 range of Electrophorus electricus at 6.20 (6.03-6.39) and 133.01 (122.40-145.50) microg l(-1), respectively and were much lower than bovine AChE at 20.94 (19.53-22.58) and 418.80 (390.60-451.60) microg l(-1), respectively. The results showed that C. batrachus have the potential to be used as a cheaper and more readily available source of AChE than other more commercially available sources.

Matched MeSH terms: Fish Proteins/isolation & purification
A preparative purification process for recombinant hepatitis B core antigen using online capture by expanded bed adsorption followed by size-exclusion chromatography

Ho CW, Tan WS, Chong FC, Ling TC, Tey BT

J Microbiol Biotechnol, 2009 Apr;19(4):416-23.
PMID: 19421000

Hepatitis B core antigen (HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus (HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the "hold-up" period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.

Matched MeSH terms: Recombinant Proteins/isolation & purification
Direct purification of recombinant hepatitis B core antigen from two different pre-conditioned unclarified Escherichia coli feedstocks via expanded bed adsorption chromatography

Ng MY, Tan WS, Abdullah N, Ling TC, Tey BT

J Chromatogr A, 2007 Nov 16;1172(1):47-56.
PMID: 17945242

Direct recovery of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli homogenates via expanded bed adsorption chromatography (EBA) has been explored in this study. Streamline DEAE was selected as the anion exchanger to recover HBcAg from heat-treated and non-heat-treated unclarified feedstocks. The use of anion-exchanger for direct extraction of proteins from unclarified feedstock is not preferred due to lack of specificity of its ligand. In this study, thermal treatment of the unclarified feedstock at 60 degrees C has resulted in 1.2- and 1.8-fold increases in yield and purity of HBcAg, respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and adsorbent. The selectivity of the anion-exchanger has also been increased as shown in the breakthrough curve obtained. Enzyme-linked immunosorbent assay showed that the antigenicity of the HBcAg from heat-treated unclarified feedstock is still preserved.

Matched MeSH terms: Recombinant Proteins/isolation & purification*
Detection of giardine gene in local isolates of Giardia duodenalis by polymerase chain reaction (PCR)

Latifah I, Teoh KY, Wan KL, Rahmah M, Normaznah Y, Rohani A

Malays J Pathol, 2005 Dec;27(2):83-9.
PMID: 17191390

Giardia duodenalis is an intestinal parasite that causes diarrhoea and malabsorption in children. The parasite also infects AIDS patients with a weak immune system. A study was carried out on six local isolates of Giardia duodenalis (110, 7304, 6304, M007, 2002 and 6307) from faeces of Orang Asli patients admitted to the Gombak Hospital. WB, a reference pathogenic strain from human and G. muris from a wild mouse, were commercially obtained from the American Type Culture Collection (ATCC). All the isolates were cultured axenically in TYI-S-33 medium. Two sets of primers were used for the techniques: primers LP1 and RP1 and primers LP2 and RP2. The sets of primers amplified giardine gene of 171 bp and 218 bp in sizes respectively. The study showed that the two sets of primers could detect G. duodenalis to the genus and species level specifically.

Matched MeSH terms: Protozoan Proteins/isolation & purification
Purification and comparison of heat shock protein 90 (Hsp90) in Candida albicans isolates from Malaysian and Iranian patients and infected mice

Khalili V, Shokri H, Khosravi AR, Akim A, Amri Saroukolaei S

J Mycol Med, 2016 Jun;26(2):94-102.
PMID: 26869383 DOI: 10.1016/j.mycmed.2015.12.007

OBJECTIVE: The purposes of this study were to purify and compare the concentration ratios of heat shock protein 90 (Hsp90) in clinical isolates of Candida albicans (C. albicans) obtained from Malaysian and Iranian patients and infected mice.
MATERIALS AND METHODS: Hsp90 was extracted using glass beads and ultracentrifugation from yeast cells and purified by ion exchange chromatography (DEAE-cellulose) and followed by affinity chromatography (hydroxyapatite). Purity of Hsp90 was controlled by SDS-PAGE and its identification was realized by immunoblotting test.
RESULTS: The graphs of ion exchange and affinity chromatography showed one peak in all C. albicans isolates obtained from both Malaysian and Iranian samples, infected mice and under high-thermal (42°C) and low-thermal (25°C) shock. In immunoblotting, the location of Hsp90 fragments was obtained around 47, 75 and 82kDa. The least average concentration ratios of Hsp90 were 0.350 and 0.240mg/g for Malaysian and Iranian isolates at 25°C, respectively, while the highest average concentration ratios of Hsp90 were 3.05 and 2.600mg/g for Malaysian and Iranian isolates at 42°C, respectively. There were differences in the ratio amount of Hsp90 between Malaysian isolates (1.01±0.07mg/g) and mice kidneys (1.23±0.28mg/g) as well as between Iranian isolates (0.70±0.19mg/g) and mice kidneys (1.00±0.28mg/g) (P<0.05).
CONCLUSION: The results showed differences in all situations tested including Iranian and Malaysian isolates, samples treated with temperatures (25°C or 42°C) and before and after infecting the mice (37°C), indicating higher virulent nature of this yeast species in high temperature in human and animal models.

Matched MeSH terms: HSP90 Heat-Shock Proteins/isolation & purification*
Enzymatic Production of Bioxylitol from Sawdust Hydrolysate: Screening of Process Parameters

Rafiqul IS, Sakinah AM, Zularisam AW

Appl Biochem Biotechnol, 2015 Jun;176(4):1071-83.
PMID: 25904039 DOI: 10.1007/s12010-015-1630-2

Xylose-rich sawdust hydrolysate can be an economic substrate for the enzymatic production of xylitol, a specialty product. It is important to identify the process factors influencing xylitol production. This research aimed to screen the parameters significantly affecting bioxylitol synthesis from wood sawdust by xylose reductase (XR). Enzymatic bioxylitol production was conducted to estimate the effect of different variables reaction time (2-18 h), temperature (20-70 °C), pH (4.0-9.0), NADPH (1.17-5.32 g/L), and enzyme concentration (2-6 %) on the yield of xylitol. Fractional factorial design was followed to identify the key process factors. The screening design identified that time, temperature, and pH are the most significant factors influencing bioxylitol production among the variables with the values of 12 h, 35 °C, and 7.0, respectively. These conditions led to a xylitol yield of 71 % (w/w). This is the first report on the statistical screening of process variables influencing enzyme-based bioxylitol production from lignocellulosic biomass.

Matched MeSH terms: Fungal Proteins/isolation & purification
Cardiotoxins from the venom of Malayan cobra (Naja naja sputatrix)

Tan NH

Arch Biochem Biophys, 1982 Oct 01;218(1):51-8.
PMID: 7149742
Matched MeSH terms: Cobra Cardiotoxin Proteins/isolation & purification*
Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption

Abdullah N, Chase HA

Biotechnol Bioeng, 2005 Nov 20;92(4):501-13.
PMID: 16080185

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.

Matched MeSH terms: Carrier Proteins/isolation & purification
Fulltext Recombinant proteins from new constructs of SAG1 and GRA7 sequences and their usefulness to detect acute toxoplasmosis

Kotresha D, Poonam D, Muhammad Hafiznur Y, Saadatnia G, Nurulhasanah O, Sabariah O, et al.

Trop Biomed, 2012 Mar;29(1):129-37.
PMID: 22543613 MyJurnal

In this study we have cloned unreported gene fragments of Toxoplasma gondii GRA7 and SAG1 and expressed the corresponding recombinant proteins, followed by evaluation of their usefulness for the serological diagnosis of toxoplasmosis. Both recombinant proteins were expressed efficiently in insoluble form, purified by single step Ni-NTA affinity chromatography and their antigenicity to detect toxoplasma specific IgG antibodies were determined by immunoblotting. A total of 60 serum samples from three groups of individuals based on their anti-toxoplasma antibody profiles were tested, namely (I) IgM+, IgG+ (n=20), (II) IgM-, IgG+ (n=20) and (III) IgM-, IgG- (n=20). Both recombinant proteins exhibited high sensitivity (100%) with sera from Group I. rGRA7 and rSAG1 reacted 40% and 80% respectively with Group II sera. The specificity of the recombinant proteins based on reactivities with Group III sera were 100% and 80% with rGRA7 and rSAG1 respectively. Thus rGRA7 was found to be better at discriminating probable acute from chronic phases of toxoplasmosis, and it also showed higher specificity.

Matched MeSH terms: Recombinant Proteins/isolation & purification
Effect of the mannose-binding Artocarpus integer lectin on the cellular proliferation of murine lymphocytes

Lim SB, Kanthimathi MS, Hashim OH

Immunol Invest, 1998 Dec;27(6):395-404.
PMID: 9845424

The effect of the mannose-binding champedak (Artocarpus integer) lectin-M on the cellular proliferation of murine lymphocytes was investigated in this study. Our data demonstrated that the lectin was the main mitogenic component in the crude extract of the champedak seeds. It stimulated the proliferation of murine T cells at an optimal concentration of 2.5 microg/ml in a 3 day culture. Lectin-M appeared to be a T-cell mitogen as it does not induce significant DNA synthesis when cultured with spleen cells from the nude mouse. In the absence of T cells, the lectin was incapable of inducing resting B cells to differentiate into immunoglobulin secreting plasma cells.

Matched MeSH terms: Carrier Proteins/isolation & purification
Response Surface Methodology Modelling of an Aqueous Two-Phase System for Purification of Protease from Penicillium candidum (PCA 1/TT031) under Solid State Fermentation and Its Biochemical Characterization

Alhelli AM, Abdul Manap MY, Mohammed AS, Mirhosseini H, Suliman E, Shad Z, et al.

Int J Mol Sci, 2016 Nov 11;17(11).
PMID: 27845736

Penicillium candidum (PCA 1/TT031) synthesizes different types of extracellular proteases. The objective of this study is to optimize polyethylene glycol (PEG)/citrate based on an aqueous two-phase system (ATPS) and Response Surface Methodology (RSM) to purify protease from Penicillium candidum (PCA 1/TT031). The effects of different PEG molecular weights (1500-10,000 g/mol), PEG concentration (9%-20%), concentrations of NaCl (0%-10%) and the citrate buffer (8%-16%) on protease were also studied. The best protease purification could be achieved under the conditions of 9.0% (w/w) PEG 8000, 5.2% NaCl, and 15.9% sodium citrate concentration, which resulted in a one-sided protease partitioning for the bottom phase with a partition coefficient of 0.2, a 6.8-fold protease purification factor, and a yield of 93%. The response surface models displayed a significant (p ≤ 0.05) response which was fit for the variables that were studied as well as a high coefficient of determination (R²). Similarly, the predicted and observed values displayed no significant (p > 0.05) differences. In addition, our enzyme characterization study revealed that Penicillium candidum (PCA 1/TT031) produced a slight neutral protease with a molecular weight between 100 and 140 kDa. The optimal activity of the purified enzyme occurred at a pH of 6.0 and at a temperature of 50 °C. The stability between different pH and temperature ranges along with the effect of chemical metal ions and inhibitors were also studied. Our results reveal that the purified enzyme could be used in the dairy industry such as in accelerated cheese ripening.

Matched MeSH terms: Fungal Proteins/isolation & purification*
Fulltext A High-Yield Two-Hour Protocol for Extraction of Human Hair Shaft Proteins

Wong SY, Lee CC, Ashrafzadeh A, Junit SM, Abrahim N, Hashim OH

PLoS One, 2016;11(10):e0164993.
PMID: 27741315 DOI: 10.1371/journal.pone.0164993

Proteome analysis of the human hair remains challenging due to the poor solubility of hair proteins and the difficulty in their extraction. In the present study, we have developed a rapid extraction protocol for hair shaft protein using alkaline-based buffer. The new protocol accelerated the procedure by reducing the extraction time from at least a day to less than two hours and showed a protein recovery of 47.3 ± 3.72%. Further analyses of the extracted protein sample through sodium dodecyl sulfate polyacrylamide gel electrophoresis and Quadrupole-time-of-flight mass spectrometry analysis unveiled a total of 60 proteins, including 25 that were not previously reported. Identification of these proteins is anticipated to be crucial in helping to understand the molecular basis of hair for potential applications in the future.

Matched MeSH terms: Proteins/isolation & purification
Danger lurking in the "unknowns": structure-to-function studies of hypothetical protein Bleg1_2437 from Bacillus lehensis G1 alkaliphile revealed an evolutionary divergent B3 metallo-beta-lactamase

Tan SH, Normi YM, Leow AT, Salleh AB, Murad AM, Mahadi NM, et al.

J. Biochem., 2017 02 01;161(2):167-186.
PMID: 28175318 DOI: 10.1093/jb/mvw058

The effectiveness of β-lactam antibiotics as chemotherapeutic agents to treat bacterial infections is gradually threatened with the emergence of antibiotic resistance mechanism among pathogenic bacteria through the production metallo-β-lactamase (MBL). In this study, we discovered a novel hypothetical protein (HP) termed Bleg1_2437 from the genome of alkaliphilic Bacillus lehensis G1 which exhibited MBL-like properties of B3 subclass; but evolutionary divergent from other circulating B3 MBLs. Domain and sequence analysis of HP Bleg1_2437 revealed that it contains highly conserved Zn2+-binding residues such as H54, H56, D58, H59, H131 and H191, important for catalysis, similar with the subclass B3 of MBL. Built 3-D Bleg1_2437 structure exhibited an αββα sandwich layer similar to the well-conserved global topology of MBL superfamily. Other features include a ceiling and floor in the model which are important for accommodation and orientation of β-lactam antibiotics docked to the protein model showed interactions at varying degrees with residues in the binding pocket of Bleg1_2437. Hydrolysis activity towards several β-lactam antibiotics was proven through an in vitro assay using purified recombinant Bleg1_2437 protein. These findings highlight the presence of a clinically important and evolutionary divergent antibiotics-degrading enzyme within the pools of uncharacterized HPs.

Matched MeSH terms: Bacterial Proteins/isolation & purification
Isolation by culture and PCR identification of LipL32 gene of pathogenic Leptospira spp. in wild rats of Kuala Lumpur

Latifah I, Abdul Halim A, Rahmat MS, Nadia MF, Ubil ZE, Asmah H, et al.

Malays J Pathol, 2017 08;39(2):161-166.
PMID: 28866698 MyJurnal

BACKGROUND: A study was conducted to confirm the status of rats as the carrier of pathogenic leptospira in Kuala Lumpur, Malaysia.
METHOD: A total of 140 urine samples were collected from trapped rats. These samples were cultured in EMJH enriched media and 18 of these samples (12.9%) were found to be positive when observed under x40 by dark field microscope. Genomic DNA was extracted from all the 18 native isolates for PCR.
RESULT: All the 18 isolates generated the expected 786 base pair band when the set of primers known to amplify LipL32 gene were utilized. These results showed that the primers were suitable to be used for the identification of pathogenic leptospira from the 18 rat samples.
CONCLUSION: The sequencing of the PCR products and BLAST analysis performed on each representative isolates confirmed the pathogenic status of all these native isolates as the LipL32 gene was detected in all the Leptospira isolates. This indicates that the rats are carriers of the pathogenic leptospira in the study area, and therefore are of public health importance.

Matched MeSH terms: Bacterial Outer Membrane Proteins/isolation & purification
Method development for analysis of proteins extracted from the leaves of Orthosiphon aristatus

Koay SY, Gam LH

J Chromatogr B Analyt Technol Biomed Life Sci, 2011 Jul 15;879(22):2179-83.
PMID: 21689998 DOI: 10.1016/j.jchromb.2011.05.041

Orthosiphon aristatus is a traditionally used medicinal plant. In order to study the proteome of the plant, we have developed a simple plant protein extraction method by direct extraction of protein using a modified 2D-gel compatible tris-sucrose buffer followed by a double TCA-acetone precipitation. This method omitted the use of toxic phenol which is widely used in the studies of plants proteins. Moreover, it shortens the lengthy extraction procedure of phenol extraction and back-extraction method and therefore reduced the extraction time (by 2h) while increased in protein yields (by 50%). Comparison of the 2D-gel images of the two extracts revealed that >60 extra protein spots were detected in the extract of our current method. The method was applied on the leaves of O. aristatus collected from six geographical areas in Malaysia. The correlation coefficient of each replicate gels from the six areas ranged from 0.70 to 0.90 indicating good reproducibility of the method.

Matched MeSH terms: Plant Proteins/isolation & purification
Fulltext Partial characterization of an enzymatic extract from Bentong ginger (Zingiber officinale var. Bentong)

Nafi' A, Ling FH, Bakar J, Ghazali HM

Molecules, 2014 Aug 15;19(8):12336-48.
PMID: 25153861 DOI: 10.3390/molecules190812336

Extraction of protease from a local ginger rhizome (Zingiber officinale var. Bentong) was carried out. The effect of extraction pH (6.4, 6.8, 7.0, 7.2, 7.6, 8.0, 8.4, and 8.8) and stabilizers (0.2% ascorbic acid, 0.2% ascorbic acid and 5 mM EDTA, or 10 mM cysteine and 5 mM EDTA) on protease activity during extraction was examined. pH 7.0 potassium phosphate buffer and 10 mM cysteine in combination with 5 mM EDTA as stabilizer were found to be the most effective conditions. The extraction procedure yielded 0.73% of Bentong ginger protease (BGP) with a specific activity of 24.8±0.2 U/mg protein. Inhibitory tests with some protease inhibitors classified the enzyme as a cysteine protease. The protease showed optimum activity at 60 °C and pH 6-8, respectively. The enzyme was completely inhibited by heavy metal cations such as Cu2+, and Hg2+. SDS stimulated the activity of enzyme, while emulsifiers (Tween 80 and Tween 20) slightly reduced its activity. The kinetic analysis showed that the protease has Km and Vmax values of 0.21 mg mL-1 and 34.48 mg mL-1 min-1, respectively. The dried enzyme retained its activity for 22 months when stored at -20 °C.

Matched MeSH terms: Plant Proteins/isolation & purification
Studies on extraction of mannanase enzyme by Aspergillus terreus SUK-1 from fermented palm kernel cake

Rashid JI, Samat N, Yusoff WM

Pak J Biol Sci, 2013 Sep 15;16(18):933-8.
PMID: 24502150

Microbial mannanases have become biotechnologically important in industry but their application is limited due to high production cost. In presents study, the extraction of mannanase from fermented Palm Kernel Cake (PKC) in the Solid State Fermentation (SSF) was optimized. Local isolate of Aspergillus terreus SUK-1 was grown on PKC in (SSF) using column bioreactor. The optimum condition were achieved after two washes of fermented PKC by adding of 10% glycerol (v/v) soaked for 10 h at the room temperature with solvent to ratio, 1:5 (w/v).

Matched MeSH terms: Bacterial Proteins/isolation & purification*
Fulltext Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

Tam YJ, Allaudin ZN, Lila MA, Bahaman AR, Tan JS, Rezaei MA

BMC Biotechnol, 2012 Oct 05;12:70.
PMID: 23039947 DOI: 10.1186/1472-6750-12-70

BACKGROUND: Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells.
RESULTS: The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively.
CONCLUSIONS: The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

Matched MeSH terms: Recombinant Proteins/isolation & purification
Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease

Fu Z, Hamid SB, Razak CN, Basri M, Salleh AB, Rahman RN

Protein Expr Purif, 2003 Mar;28(1):63-8.
PMID: 12651108

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium. The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction. The recombinant F1 protease was efficiently excreted into the culture medium using E. coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs. Both vectors contain the E. coli lac promoter-operator system. In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium. This opens the way for the large-scale production of this protease in E. coli. The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).

Matched MeSH terms: Recombinant Proteins/isolation & purification

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