Winston Su - Academia.edu (original) (raw)

Papers by Winston Su

Microbial cell factories, Jan 25, 2005

Fluorescent proteins are genetically encoded, highly versatile reporters useful for monitoring va... more Fluorescent proteins are genetically encoded, highly versatile reporters useful for monitoring various aspects of recombinant protein production. In addition to the widely popular green fluorescent protein (GFP) from Aequorea victoria, a variety of other fluorescent proteins have been discovered that display a wide range of spectral properties. Synthetic variants have also been developed to overcome limitations associated with their wild-type counterparts. Having a large repertoire of fluorescent proteins with diverse traits opens new opportunities for rapid monitoring and optimization of recombinant protein production.

A modified culture protocol has been developed for the induction of somatic embryogenesis in Azad... more A modified culture protocol has been developed for the induction of somatic embryogenesis in Azadirachta indica (neem). Embryogenic calluses were initiated from cotyledons or hypocotyls using a Murashige and Skoog (MS) agar medium supplemented with 0.5 mg l 1-napthaleneacetic acid (NAA), 1 mg l 1 6-benzylaminopurine (BA), 1 g l 1 casein hydrolysate, and 50 g l 1 sucrose. The calluses, when transferred to a liquid medium similar to the agar medium but with NAA replaced by 0.5 mg l 1 indole-3-acetic acid (IAA), formed globular structures which further developed a rudimentary root, after 4 to 5 weeks incubation. Subsequently, these highly differentiated tissues when transferred into a hormone-free MS medium containing 1 g l 1 casein hydrolysate and 50 g l 1 sucrose, active embryo masses started to appear after 1 to 2 weeks. The embryo production was found to improve more than 2 fold by adding 0.2 mg l 1 zeatin to the medium.

Scientific Reports, 2015

Being able to coordinate co-expression of multiple proteins is necessary for a variety of importa... more Being able to coordinate co-expression of multiple proteins is necessary for a variety of important applications such as assembly of protein complexes, trait stacking and metabolic engineering. Currently only few options are available for multiple recombinant protein co-expression and most of them are not applicable to both prokaryotic and eukaryotic hosts. Here, we report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expression of multiple proteins. The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linker to mediate efficient release of the flanking proteins via autocatalytic cleavage. Essentially complete release of constituent proteins, GFP and RFP (mCherry), from a polyprotein precursor, in bacterial, mammalian and plant hosts was demonstrated. In addition, successful co-expressi...

Biochemical Engineering for 2001, 1992

Bubble-free aeration using immersed porous membranes in bioreactors has been proposed for more th... more Bubble-free aeration using immersed porous membranes in bioreactors has been proposed for more than a decade. Membrane aeration systems sustain high gas transfer capabilities under high culture viscosity due to the fact that they have constant gas/liquid interfacial area. This is a very desirable feature for aerating plant cell cultures since plant cell cultures can be very viscous at high cell densities. Membrane aeration can also eliminate foaming and cell floatation and, in turn, the undesirable wall growth and culture deterioration. In addition, cellular hydrodynamic shear damage due to bubble breakup, which is believed to be the major contributor to death under mild agitation in sparged, mechanically agitated reactors [1], can be avoided when membrane aeration is used.

2005 Tampa, FL July 17-20, 2005, 2005

ABSTRACT

Green fluorescence protein (GFP) has become a widely used reporter in many areas of life science.... more Green fluorescence protein (GFP) has become a widely used reporter in many areas of life science. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. GFP itself has been purified from recombinant organisms by several methods, often involving unfavorable conditions (e.g., use of organic solvents and/or low pH) that may be destabilizing to some proteins. In this study, we have developed a general recovery scheme that entails a simple three-step purification procedure for GFP fusion proteins produced in tobacco suspension cells, with the intent of maximizing purity and yield under gentle conditions so as to maintain the integrity of the fusion partner. Ammonium sulfate treatment at 30% (v/v) precipitated particulate matter and removed aggregated material while simultaneously maintaining GFP solubility and increasing hydrophobicity. Hydrophobic interaction chromatography was then performed to eliminate the majority of background proteins while eluting GFP and fusions in a low ionic buffer suitable to be directly applied to an ion-exchange column as the final step. Three intracellular proteins, secreted alkaline phosphatase (SEAP), and granulocyte-macrophage colony-stimulating factor (GMCSF), each fused to GFP, as well as GFP itself, were recovered with yields exceeding 70% and purity levels over 80%. This purification scheme exploits the hydrophobic nature of GFP while maintaining a gentle environment for labile fusion partners. Although some optimization may be required, we believe this scheme may serve as a benchmark for purifying other GFP fusion proteins.

Transgenic Research, 2005

Proceedings of the National Academy of Sciences, 2010

Endoplasmic reticulum (ER)-associated degradation (ERAD) is an integral part of the ER quality-co... more Endoplasmic reticulum (ER)-associated degradation (ERAD) is an integral part of the ER quality-control system that removes toxic misfolded proteins via ubiquitin/proteasome-mediated degradation. Most of our knowledge on ERAD comes from biochemical and genetic studies in yeast and mammalian cells. Although ERAD is known to operate in plant cells, little is known about its molecular components and its biochemical mechanism. A genetic screen for suppressors of the Arabidopsis bri1-9 , a weak dwarf mutant caused by ER retention of a structurally defective yet biochemically competent brassinosteroid (BR) receptor BRI1, resulted in identification of the EMS-mutagenized bri1 suppressor 5 ( EBS5 ) gene that encodes an Arabidopsis homolog of the yeast Hrd3/mammlian Sel1L protein known to be involved in ERAD. Loss-of-function ebs5 mutations block the ERAD of bri1-9 and bri1-5, another ER-retained BR receptor. We showed that EBS5 complemented the ERAD defect of the yeast Δhrd3 mutant and inter...

Plant Biotechnology Journal, 2011

Journal of Microbiological Methods, 2011

Rapid concentration of bacterial targets from dilute solutions to improve subsequent PCR detectio... more Rapid concentration of bacterial targets from dilute solutions to improve subsequent PCR detection is investigated in this study. Submicron (average size 500 nm) superparamagnetic anion-exchangers (SiMAG-DEAE) were used successfully to concentrate target bacteria from very dilute solutions. A mass-balance model predicted that for Escherichia coli, the extent of cell concentrating increases almost linearly with increasing sample/SiMAG volume ratio up to about 2000, accompanied by only a slight decrease in the capture efficiency (b 10%). Our experimental data generally support this analysis in that the SiMAG beads concentrated bacterial targets by two to three orders of magnitude using a sample/bead volume ratio of about 1000, and lowered the PCR detection limit to a level of 10 2 CFU/mL, from 10 4 to 10 5 CFU/mL without concentrating. Several target bacteria can be concentrated concurrently and detected via multiplex PCR, as illustrated using E. coli and Agrobacterium tumefaciens as model bacteria. Finally, concentration and detection of bacteria in fresh produce samples were demonstrated. The integration of submicron magnetic ion exchangers and PCR detection provides an appealing alternative to immunomagnetic separation/PCR in improving pathogen detection.

Journal of Biotechnology, 2010

Glucoamylase and alpha-amylase co-immobilized with gamma ferric oxide powders in chitosan beads f... more Glucoamylase and alpha-amylase co-immobilized with gamma ferric oxide powders in chitosan beads for consecutive starch liquefaction and saccharification under different magnetic fields was investigated. The chitosan concentration in the beads was found to greatly affect the immobilized enzyme performance. Superior immobilization efficiency and enzyme stability were noted when 2% instead of 4% chitosan was utilized. Using confocal microscopy and scanning electron microscopy, the beads with 2% chitosan were seen to exhibit a more rugged surface topology with more macropores and accommodate more protein near the external surface than with the 4% chitosan beads. An optimum loading ratio between alpha-amylase and glucoamylase exists that gives the highest glucose production, and this ratio varies with the size of the beads. The inclusion of the gamma ferric oxide powders renders the beads magnetically anisotropic and causes them to tumble under a single-phase alternating magnetic field, resulted in increased overall reaction rates. When exposed to a three-phase alternating magnetic field, these beads were stirred vigorously, also leading to enhanced reaction rates. The use of multi-enzyme co-immobilization in magnetic anisotropic chitosan beads may be extended to other practical applications that involve coordinated enzymatic reactions of macromolecular substrates.

Journal of Biotechnology, 1997

In this study, extracellular enzyme production by fungal pellets cultured in a novel continuous p... more In this study, extracellular enzyme production by fungal pellets cultured in a novel continuous perfusion bioreactor is investigated. Cell retention during perfusion culture is achieved by incorporating an internal settling zone into an external-loop airlift bioreactor. Production of an extracellular enzyme, acid phosphatase, by the filamentous fungus Neurospora crassa was chosen as a model system. In order to control culture morphology to allow effective long-term perfusion culture, an anionic polymer Carbopol (carboxypolymethylene) at 0.1% was added to the culture medium to promote growth in a more dispersed form. The bioreactor has shown a high pellet retention efficiency over a wide range of medium perfusion rates. The fungal pellets were successfully cultivated in the bioreactor for over 30 days. By operating the bioreactor under phosphate limitation, and by a step-wise increase of the perfusion rate from 0.5 to 1 d −1 , a steady state phosphatase volumetric productivity of ca. 900 U l − 1 d − 1 was reached while cell dry weight was maintained at over 4 g l −1 .

Journal of Biotechnology, 2003

Journal of Biotechnology, 1996

Journal of Bioscience and Bioengineering, 2003

Culture perfusion is widely practiced in mammalian cell processes to enhance secreted antibody pr... more Culture perfusion is widely practiced in mammalian cell processes to enhance secreted antibody production. Here, we report the development of an efficient continuous perfusion process for the cultivation of plant cell suspensions. The key to this process is a perfusion bioreactor that incorporates an annular settling zone into a stirred-tank bioreactor to achieve continuous cell/medium separation via gravitational sedimentation. From washout experiments, we found that under typical operating conditions (e.g., 200 rpm and 0.3 vvm) the liquid phase in the entire perfusion bioreactor was homogeneous despite the presence of the cylindrical baffle. Using secreted acid phosphatase (APase) produced in Anchusa officinalis cell culture as a model we have studied the perfusion cultures under complete or partial cell retention. The perfusion culture was operated under phosphate limitation to stimulate APase production. Successful operation of the perfusion process over four weeks has been achieved in this work. When A. officinalis cells were grown in the perfusion reactor and perfused at up to 0.4 vvd with complete cell retention, a cell dry weight exceeding 20 g/l could be achieved while secreted APase productivity leveled off at approximately 300 units/l/d. The culture became extremely dense with the maximum packed cell volume (PCV) surpassing 70%. In comparison, the maximum cell dry weight and overall secreted APase productivity in a typical batch culture were 10-12 g/l and 100-150 units/l/d, respectively. Operation of the perfusion culture under extremely high PCV for a prolonged period, however, led to declined oxygen uptake and reduced viability. Subsequently, cell removal via a bleed stream at up to 0.11 vvd was tested and shown to stabilize the culture at a PCV below 60%. With culture bleeding, both specific oxygen uptake rate and viability were shown to increase. This also led to a higher cell dry weight exceeding 25 g/l, and further improvement of secreted APase productivity that reached a plateau fluctuating around 490 units/l/d.

Enzyme and Microbial Technology, 2001

In this study, expression of green fluorescence protein (GFP) on the external surface of Escheric... more In this study, expression of green fluorescence protein (GFP) on the external surface of Escherichia coli was achieved by construction of a fusion protein between Lpp-OmpA hybrid and a GFP variant, GFPmut2. The GFP was fused in frame to the carboxyl-terminus of Lpp-OmpA fusion previously shown to direct various other heterologous proteins to E. coli cell surface. Western blot analysis of membrane fractions identified the Lpp-OmpA-GFP fusion protein with the expected size (43 kDa). Immunofluorescence microscopy, immunoelectron microscopy, protease and extracellular pH sensitivity assays further confirmed that GFP is anchored on the outer membrane. The GFP displayed on the E. coli outer surface retained its fluorescence and was not susceptible to the indigenous outer membrane protease OmpT even though there are two putative OmpT proteolytic sites present in GFP. Optimization of the expression conditions was conducted using fluorometry, eliminating cumbersome immuno-labeling procedures. Surface-displayed GFP could be used in a variety of applications including screening of polypeptide libraries, development of live vaccines, construction of whole cell allosteric biosensors, and signal transduction studies.

Biotechnology Progress, 1992

In this paper, a theoretical analysis of oxygen transport across the tubular microporous membrane... more In this paper, a theoretical analysis of oxygen transport across the tubular microporous membrane is described. This analysis has provided some insight into the optimal design of the membrane aerator. It was found in this study, a t fixed inlet pressure, that the overall membrane oxygen transfer rate increases with increased tubing length only up to a certain length, i.e., the "critical length". When a large membrane surface area is required, the fiber should be divided into parallel segments to increase the overall oxygen transfer rate. A manifold or a gas distributor can then be used to distribute gas into segments of tubing. The length of each segment cannot exceed the critical length. In addition, shorter tube segments should give a higher oxygen transfer rate per unit tube length; however, this advantage is counterbalanced by the fact that gas distribution into huge numbers of parallel tubings may not be uniform.

Biotechnology Letters, 1993

High cell density and rosmarinic acid (RA) productivity have been achieved by applying periodic c... more High cell density and rosmarinic acid (RA) productivity have been achieved by applying periodic culture perfusion to the Anchusa officinalis cell suspension. In this study, the effect of inoculum size on cell growth and RA productivity in the perfused Anchusa culture was investigated. Experimental results showed that RA productivity increased with the inoculum size, up to 4 g dry weight/L. Further increases in the inoculum size did not yield a higher RA productivity regardless of culture perfusion. Moreover, the maximum cell concentration was not affected by the inoculum sizes, from 1 to 11 g dry weight/L. Cell crowding, indicated by high culture packed cell volumes, is believed to be the predominant cause of low productivity in perfused cultures with high inoculum sizes.

Biotechnology Letters, 1991

In this study, a perfusion fermentation of Anchusa officinalis was carried out in a stirred tank ... more In this study, a perfusion fermentation of Anchusa officinalis was carried out in a stirred tank bioreactor integrated with art internal cross-flow f'dter. Bubble-free aeration via microporous membrane fibers was used to provide oxygen. A two-stage culture was successfully conducted in this reactor without f'dter fouling. In a 17 day fermentation, a cell density of 26 g dw/1 and a rosmafinic acid productivity of 94 rag/l-day were achieved. This productivity is three times that obtained in a batch culture.