Microfiltration: Effect of channel diameter on limiting flux and serum protein removal.

Our objective was to determine the limiting flux and serum protein (SP) removal at 8, 9 and 10% true protein (TP) in the retentate recirculation loop using 0.1-µm ceramic graded permeability (GP) microfiltration (MF) membranes with 3mm channel diameters (CD). An additional objective was to compare the limiting flux and SP removal between 0.1-µm ceramic GP membranes with 3mm CD and previous research using 4-mm CD membranes. The MF system was operated at 50°C, using a diluted milk protein concentrate with 85% protein on a total solids basis (MPC85) as the MF feed. The limiting flux for the MF of diluted MPC85 was determined at 8, 9, and 10% TP concentration in the recirculation loop. The experiment using the 3-mm CD membranes was replicated 3 times for a total of 9 runs. On the morning of each run MPC85 was diluted with reverse osmosis water to a MF feed TP concentration of 5.4%. In all runs the starting flux was 55 kg/m2 per hour, the flux was then increased in steps until the limiting flux was reached. For the 3-mm CD membranes, the limiting flux was 128±0.3, 109±4, and 97±0.5 kg/m2 per hour at recirculation loop TP concentrations of 8.1±0.07, 9.2±0.04, and 10.2±0.03%, respectively. For the 3-mm CD membranes, increasing the flux from the starting to the limiting flux decreased the SP removal factor from 0.72±0.02 to 0.67±0.01; however, no difference in SP removal factor among the target recirculation loop TP concentrations was detected. The limiting flux at each recirculation loop target TP concentration was lower for the 3- compared with the 4-mm CD membranes. The differences in limiting fluxes between the 3- and 4-mm CD membranes were explained in part by the difference in cross-flow velocity (5.5±0.03 and 7.0±0.03 m/s for the 3- and 4-mm CD membranes, respectively). The SP removal factor was also lower for the 3- compared with the 4-mm CD membranes, indicating that more membrane fouling may have occurred in the 3- versus 4-mm CD membranes.

Microfiltration: Effect of retentate protein concentration on limiting flux and serum protein removal with 4-mm-channel ceramic microfiltration membranes.

The objective of our study was to determine if the limiting flux and serum protein (SP) removal were different at 8, 9, or 10% true protein (TP) in the microfiltration (MF) retentate recirculation loop using 0.1-µm ceramic graded permeability membranes with 4-mm-channel diameters operated at 50 °C using a diluted milk protein concentrate with 85% protein on a total solids basis (MPC85) as the MF feed. The limiting flux for the MF of diluted MPC85 was determined at 3 TP concentrations in the recirculation loop (8, 9, and 10%). The experiment was replicated 3 times for a total of 9 runs. On the morning of each run, MPC85 was diluted with reverse osmosis water to an MF feed TP concentration of 5.4%. In all runs, the starting flux was 55 kg/m(2) per hour, the flux was increased in steps until the limiting flux was reached. The minimum flux increase was 10 kg/m(2) per hour. The limiting flux decreased as TP concentration in the recirculation loop increased. The limiting flux was 154 ± 0.3, 133 ± 0.7, and 117 ± 3.3 kg/m(2) per hour at recirculation loop TP concentrations of 8.2 ± 0.07, 9.2 ± 0.04, and 10.2 ± 0.09%, respectively. No effect of recirculation loop TP concentration on the SP removal factor was detected. However, the SP removal factor decreased from 0.80 ± 0.02 to 0.75 ± 0.02 as flux was increased from the starting flux of 55 kg/m(2) per hour to the limiting flux, with a similar decrease seen at all recirculation loop TP concentrations.

Microfiltration of skim milk and modified skim milk using a 0.1-µm ceramic uniform transmembrane pressure system at temperatures of 50, 55, 60, and 65°C.

Increasing the temperature of microfiltration (MF) to >50°C may allow for operation at higher fluxes and reduce the bacterial growth during MF. However, there is a concern that operating at higher temperatures could cause calcium phosphate precipitation that would lead to membrane fouling. Our objective was to determine the effect of operating a 0.1-µm ceramic uniform transmembrane pressure MF unit at temperatures of 50, 55, 60, and 65°C on membrane fouling and serum protein (SP) removal from skim milk with and without removal of low-molecular-weight soluble milk components by ultrafiltration (UF) before MF at a flux of 54kg/m(2) per hour. For each replicate, 1,000kg of pasteurized skim milk was split into 2 batches. One batch was ultrafiltered (with diafiltration) to remove an average of 89±2% of the lactose and a percentage of the soluble calcium and phosphorus. The retentate from UF was diluted back to the protein concentration of skim milk, creating the diluted UF retentate (DUR). On subsequent days, both the DUR and skim milk were run on the MF unit with the flux maintained at 54kg/m(2) per hour and a concentration factor of 3× and the system run in recycle mode. The temperature of MF was increased in 5°C steps from 50 to 65°C, with a 1-h stabilization period after each increase. During the run, transmembrane pressure was monitored and permeate and retentate samples were taken and analyzed to determine if any changes in SP, calcium, or phosphorus passage through the membrane occurred. Increasing temperature of MF from 50 to 65°C at a flux of 54kg/m(2) per hour did not produce a large increase in membrane fouling when using either skim milk or a DUR as the MF feed type as measured by changes in transmembrane pressure. Increasing the temperature to 65°C only caused a slight reduction in calcium concentration in the permeate (11±3%) that was similar between the 2MF feed types. Increasing processing temperature reduced the percentage of SP removal by the process, but the increased temperature also caused a decrease in casein contamination in the permeate with no evidence of membrane fouling.

From nucleoporins to nuclear pore complexes.

One of the largest supramolecular assemblies in the eukaryotic cell, the nuclear pore complex, is now being dissected into its numerous molecular constituents. The combined use of biochemistry and genetics in yeast has made this rapid development possible. Although less is known about vertebrate nucleoporins, the first clues are now emerging about their in vivo function also. Much remains to be learned about nuclear pore complex assembly and function, however.

Nuclear transport.

A striking property of nuclear pore complexes is their ability to mediate bi-directional nucleocytoplasmic traffic of proteins and RNAs. In the past year, several new nuclear pore proteins have been identified, but their precise functions remain to be established. Cytosolic factors responsible for the recognition and docking of substrates for nuclear transport are also being characterized. It appears that different factors are required for the import of karyophilic proteins versus small nuclear ribonucleoprotein particles. Furthermore, the GTPase Ran/TC4 has been shown to play a key role in translocation across the nuclear pore complex. Specific RNAs require different sets of factors for their export from the nucleus, although a common export route appears to be utilized by different RNA species. In contrast, nuclear retention has been found to have an influence in controlling the rate of protein exit from the nucleus.

Processing factors that influence casein and serum protein separation by microfiltration.

Our objective was to demonstrate the effect of various processing factors on the performance of a microfiltration system designed to process skim milk and separate casein (CN) from serum proteins (SP). A mathematical model of a skim milk microfiltration process was developed with 3 stages plus an additional fourth finishing stage to standardize the retentate to 9% true protein (TP) and allow calculation of yield of a liquid 9% TP micellar CN concentrate (MCC) and milk SP isolate (MSPI; 90% SP on a dry basis). The model was used to predict the effect of 5 factors: 1) skim milk composition, 2) heat treatment of skim milk, 3) concentration factor (CF) and diafiltration factor (DF), 4) control of CF and DF, and 5) SP rejection by the membrane on retentate and permeate composition, SP removal, and MCC and MSPI yield. When skim milk TP concentration increased from 3.2 to 3.8%, the TP concentration in the third stage retentate increased from 7.92 to 9.40%, the yield of MCC from 1,000 kg of skim milk increased from 293 to 348 kg, and the yield of MSPI increased from 6.24 to 7.38 kg. Increased heat treatment (72.9 to 85.2°C) of skim milk caused the apparent CN as a percentage of TP content of skim milk as measured by Kjeldahl analysis to increase from 81.97 to 85.94% and the yield of MSPI decreased from 6.24 to 4.86 kg, whereas the third stage cumulative percentage SP removal decreased from 96.96 to 70.08%. A CF and DF of 2× gave a third stage retentate TP concentration of 5.38% compared with 13.13% for a CF and DF of 5×, with the third stage cumulative SP removal increasing from 88.66 to 99.47%. Variation in control of the balance between CF and DF (instead of an equal CF and DF) caused either a progressive increase or decrease in TP concentration in the retentate across stages depending on whether CF was greater than DF (increasing TP in retentate) or CF was less than DF (decreasing TP in retentate). An increased rejection of SP by the membrane from an SP removal factor of 1 to 0.6 caused a reduction in MSPI yield from 6.24 to 5.19 kg/1,000 kg of skim milk, and third stage cumulative SP removal decreased from 96.96 to 79.74%. Within the ranges of the 5 factors studied, the TP content of the third stage retentate was most strongly affected by the target CF and DF and variation in skim milk composition. Cumulative percentage SP removal was most strongly affected by the heat treatment of skim milk, the SP removal factor, and the target CF and DF. The MCC yield was most strongly affected by initial skim milk composition. Yield of MSPI was strongly affected by skim milk composition, whereas the heat treatment of milk and SP removal factor also had a large effect.

Micellar casein concentrate production with a 3X, 3-stage, uniform transmembrane pressure ceramic membrane process at 50°C.

The production of serum protein (SP) and micellar casein from skim milk can be accomplished using microfiltration (MF). Potential commercial applications exist for both SP and micellar casein. Our research objective was to determine the total SP removal and SP removal for each stage, and the composition of retentates and permeates, for a 3×, continuous bleed-and-feed, 3-stage, uniform transmembrane pressure (UTP) system with 0.1-µm ceramic membranes, when processing pasteurized skim milk at 50°C with 2 stages of water diafiltration. For each of 4 replicates, about 1,100 kg of skim milk was pasteurized (72°C, 16s) and processed at 3× through the UTP MF system. Retentate from stage 1 was cooled to <4°C and stored until the next processing day, when it was diluted with reverse osmosis water back to a 1× concentration and again processed through the MF system (stage 2) to a 3× concentration. The retentate from stage 2 was stored at <4°C, and, on the next processing day, was diluted with reverse osmosis water back to a 1× concentration, before running through the MF system at 3× for a total of 3 stages. The retentate and permeate from each stage were analyzed for total nitrogen, noncasein nitrogen, and nonprotein nitrogen using Kjeldahl methods; sodium dodecyl sulfate-PAGE analysis was also performed on the retentates from each stage. Theoretically, a 3-stage, 3× MF process could remove 97% of the SP from skim milk, with a cumulative SP removal of 68 and 90% after the first and second stages, respectively. The cumulative SP removal using a 3-stage, 3× MF process with a UTP system with 0.01-µm ceramic membranes in this experiment was 64.8 ± 0.8, 87.8 ± 1.6, and 98.3 ± 2.3% for the first, second, and third stages, respectively, when calculated using the mass of SP removed in the permeate of each stage. Various methods of calculation of SP removal were evaluated. Given the analytical limitations in the various methods for measuring SP removal, calculation of SP removal based on the mass of SP in the skim milk (determined by Kjeldahl) and the mass SP present in all of the permeate produced by the process (determined by Kjeldahl) provided the best estimate of SP removal for an MF process.

Targeting of a cytosolic protein to the nuclear periphery.

The yeast nuclear envelope protein NSP1 is located at the nuclear pores and mediates its essential function via the carboxy-terminal domain. The passenger protein, cytosolic dihydrofolate reductase from mouse, was fused to the 220 residue long NSP1 carboxy-terminal domain. When expressed in yeast, this chimeric protein was tightly associated with nuclear structures and was localized at the nuclear periphery very similar to authentic NSP1. Furthermore, the DHFR-C-NSP1 fusion protein was able to complement a yeast mutant lacking a functional NSP1 gene showing that DHFR-C-NSP1 fulfils the same basic function as compared to the endogenous NSP1 protein. These data also show that the NSP1 protein is composed of separate functional moieties: a carboxy-terminal domain that is sufficient to mediate the association with the nuclear periphery and an amino-terminal and middle repetitive domain with an as yet unknown function. It is suggested that heptad repeats found in the NSP1 carboxy-terminal domain, which are similar to those found in intermediate filament proteins, are crucial for mediating the association with the nuclear pores.

Biogenesis of the signal recognition particle (SRP) involves import of SRP proteins into the nucleolus, assembly with the SRP-RNA, and Xpo1p-mediated export.

The signal recognition particle (SRP) targets nascent secretory proteins to the ER, but how and where the SRP assembles is largely unknown. Here we analyze the biogenesis of yeast SRP, which consists of an RNA molecule (scR1) and six proteins, by localizing all its components. Although scR1 is cytoplasmic in wild-type cells, nuclear localization was observed in cells lacking any one of the four SRP "core proteins" Srp14p, Srp21p, Srp68p, or Srp72p. Consistently, a major nucleolar pool was detected for these proteins. Sec65p, on the other hand, was found in both the nucleoplasm and the nucleolus, whereas Srp54p was predominantly cytoplasmic. Import of the core proteins into the nucleolus requires the ribosomal protein import receptors Pse1p and Kap123p/Yrb4p, which might, thus, constitute a nucleolar import pathway. Nuclear export of scR1 is mediated by the nuclear export signal receptor Xpo1p, is distinct from mRNA transport, and requires, as evidenced by the nucleolar accumulation of scR1 in a dis3/rrp44 exosome component mutant, an intact scR1 3' end. A subset of nucleoporins, including Nsp1p and Nup159p (Rat7p), are also necessary for efficient translocation of scR1 from the nucleus to the cytoplasm. We propose that assembly of the SRP requires import of all SRP core proteins into the nucleolus, where they assemble into a pre-SRP with scR1. This particle can then be targeted to the nuclear pores and is subsequently exported to the cytoplasm in an Xpo1p-dependent way.

Binding of the Mex67p/Mtr2p heterodimer to FXFG, GLFG, and FG repeat nucleoporins is essential for nuclear mRNA export.

It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore. Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins. For this interaction, complex formation between Mex67p and Mtr2p has to occur. MEX67 and MTR2 also genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p. These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore.