Downstream protein separation by surfactant precipitation: a review

In a conventional protein downstream processing (DSP) scheme, chromatography is the single most expensive step. Despite being highly effective, it often has a low process throughput due to its semibatch nature, sometimes with nonreproducible results and relatively complex process development. Hence, more work is required to develop alternative purification methods that are more cost-effective, but exhibiting nearly comparable performance. In recent years, surfactant precipitation has been heralded as a promising new method for primary protein recovery that meets these criteria and is a simple and cost-effective method that purifies and concentrates. The method requires the direct addition of a surfactant to a complex solution (e.g. a fermentation broth) containing the protein of interest, where the final surfactant concentration is maintained below its critical micelle concentration (CMC) in order to allow for electrostatic and hydrophobic interactions between the surfactant and the target protein. An insoluble (hydrophobic) protein–surfactant complex is formed and backextraction of the target protein from the precipitate into a new aqueous phase is then carried out using either solvent extraction, or addition of a counter-ionic surfactant. Importantly, as highlighted by past researchers, the recovered proteins maintain their activity and structural integrity, as determined by circular dichroism (CD). In this review, various aspects of surfactant precipitation with respect to its general methodology and process mechanism, system parameters influencing performance, protein recovery, process selectivity and process advantages will be highlighted. Moreover, comparisons will be made to reverse micellar extraction, and the current drawbacks/challenges of surfactant precipitation will also be discussed. Finally, promising directions of future work with this separation technique will be highlighted.     

Optimization of parameters for improvement of phenol degradation by Rhodococcus UKMP-5M using response surface methodology

Phenol is a toxic compound and is one of the major pollutants contained in the waste water from petroleum and its downstream industries. Response surface methodology (RSM) was used to optimize medium composition and culture condition for enhancement of growth of Rhodococcus UKMP-5M and phenol degradation rate in shake flask cultures. Phenol and (NH₄)₂SO₄ concentrations as well as temperature were the most significant factors that influenced growth and phenol degradation. Central composite design (CCD) was used for optimization of these parameters with growth, and degradation rates were used as the responses. Cultivation with 0.5 g/L phenol and 0.3 g/L (NH₄)₂SO₄ and incubation at 36 °C greatly enhanced growth of Rhodococcus UKMP-5M, where the final cell concentration increased from 0.117 g/L to 0.376 g/L. On the other hand, the degradation rate was greatly increased in cultivation with 0.7 g/L phenol and 0.4 g/L (NH₄)₂SO₄ and incubation at 37 °C. In this cultivation, the time taken to degrade 1 g/L phenol in the culture was reduced from 48 h to 27 h. The model for both responses was found significant and the predicted values were found to be in a good agreement with experimental values and subsequently validated. Increases in phenol degradation rate during Rhodococcus UKMP-5M cultivation corresponded well with increasing phenol hydroxylase activity.     

Common Oncogenic Mutations Are Infrequent in Oral Squamous Cell Carcinoma of Asian Origin

Objectives

The frequency of common oncogenic mutations and TP53 was determined in Asian oral squamous cell carcinoma (OSCC).

Materials and Methods

The OncoCarta^™ panel v1.0 assay was used to characterize oncogenic mutations. In addition, exons 4-11 of the TP53 gene were sequenced. Statistical analyses were conducted to identify associations between mutations and selected clinico-pathological characteristics and risk habits.

Results

Oncogenic mutations were detected in PIK3CA (5.7%) and HRAS (2.4%). Mutations in TP53 were observed in 27.7% (31/112) of the OSCC specimens. Oncogenic mutations were found more frequently in non-smokers (p = 0.049) and TP53 truncating mutations were more common in patients with no risk habits (p = 0.019). Patients with mutations had worse overall survival compared to those with absence of mutations; and patients who harbored DNA binding domain (DBD) and L2/L3/LSH mutations showed a worse survival probability compared to those patients with wild type TP53. The majority of the oncogenic and TP53 mutations were G:C > A:T and A:T > G:C base transitions, regardless of the different risk habits.

Conclusion

Hotspot oncogenic mutations which are frequently present in common solid tumors are exceedingly rare in OSCC. Despite differences in risk habit exposure, the mutation frequency of PIK3CA and HRAS in Asian OSCC were similar to that reported in OSCC among Caucasians, whereas TP53 mutations rates were significantly lower. The lack of actionable hotspot mutations argue strongly for the need to comprehensively characterize gene mutations associated with OSCC for the development of new diagnostic and therapeutic tools.     

Purification of filamentous bacteriophage M13 by expanded bed anion exchange chromatography

In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (Ho = 15 cm) of STREAMLINE DEAE (rho = 1.2 g/cm3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.     

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production ofEscherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h⁻¹. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h⁻¹) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C.     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.     

Anaerobic fermentation of gelatinized sago starch-derived sugars to acetone—1-butanol—Ethanol solvent byClostridium acetobutylicum

A study of the kinetics and performance of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymic hydrolysis of sago starch byClostridium acetobutylicum showed that the use of 30 g/L gelatinized sago starch as the sole carbon source produced 11.2 g/L total solvent,i.e. 1.5–2 times more than with pure maltose or glucose used as carbon sources. Enzymic pretreatment of gelatinized sago starch yielding maltose and glucose hydrolyzates prior to the fermentation did not improve solvent production as compared to direct fermentation of gelatinized sago starch. The solvent yield of direct gelatinized sago starch fermentation depended on the activity and stability of amylolytic enzymes produced during the fermentation. The pH optima for α-amylase and glucoamylase were found to be at 5.3 and 4.0–4.4, respectively. α-Amylase showed a broad pH stability profile, retaining more than 80% of its maximum activity at pH 3.0–8.0 after a 1-d incubation at 37°C. SinceC. acetobutylicum α-amylase has a high activity and stability at low pH, this strain can potentially be employed in a one-step direct solvent-yielding fermentation of sago starch. However, theC. acetobutylicum glucoamylase was only stable at pH 4–5, maintaining more than 90% of its maximum activity after a 1-d incubation at 37°C.     

Characterization of Salmonella spp. isolated from patients below 3 years old with acute diarrhoea

Fifteen strains of Salmonella were isolated from children with clinically diagnosed diarrhoea aged below 3 years old, who had been admitted to K7 ward, Pediatric Institute, Kuala Lumpur Hospital. The isolates were tested for their susceptibility to a range of antimicrobial agents, and typed by serological tests and randomly amplified polymorphic DNA (RAPD) fingerprinting. All the strains had a similar pattern of antimicrobial susceptibility, where they were susceptible to a wide range of antimicrobial agents. The serological test has typed them into three serovars, which were identified as Salmonella enterica ser. Akanji, Salmonella enterica ser. Hindmarch and Salmonella enterica ser. Richmond. In contrast, the RAPD fingerprinting classed them into two major clusters, cluster 1 consisting of 12 strains of Salmonella and cluster 2 consisting of three strains of Salmonella.     

Novel approaches to manipulating foetal cells in the maternal circulation for non-invasive prenatal diagnosis of the unborn child

Due to the risks to the foetus with invasive prenatal diagnosis, non-invasive prenatal diagnosis (NIPD) is gaining tremendous interest but no reliable method that can be widely used has been developed to date. Manipulation of foetal cells and foetal cell-free genetic material in the maternal blood are two promising approaches being researched. The manipulation of foetal cells in the maternal circulation is more popular as it can provide complete genetic information of the foetus particularly the diagnosis of aneuploidies. However, the foetal cell numbers in the maternal circulation are small and their enrichment and ex vivo culture remain two major challenges for NIPD. Primitive foetal erythroblasts (pFEs) have been considered as a good potential candidate for early first trimester NIPD but their nature, properties and manipulation to provide adequate cell numbers remain a challenging task and several approaches need to be meticulously evaluated. In this review we describe the current status of NIPD and suggest some novel approaches in manipulating pFEs for future clinical application of NIPD. These novel approaches include (1) understanding the pFE enucleation process, (2) enriching pFE numbers by individual pick-up of pFEs from maternal blood using micromanipulation and microdroplet culture, (3) expansion of pFEs using mitogens and (4) decondensation of the pFE nucleus with histone deacetylase (HDAC) inhibitors followed by reprogramming using gene delivery protocols with/without small reprogramming molecules to improve reprogrammed pFE proliferation rates for successful NIPD.