Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.

To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and ß-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5-6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean.

Influence of bovine serum albumin on the secondary structure of interferon alpha 2b as determined by far UV circular dichroism spectropolarimetry.

Many therapeutic biologics are formulated with excipients, including the protein excipient human serum albumin (HSA), to increase stability and prevent protein aggregation and adsorption onto glass vials. One biologic formulated with albumin is interferon alpha-2b (IFN alpha-2b). As is the case with other therapeutic biologics, the increased structural complexity of IFN alpha-2b compared to a small molecule drug requires that both the correct chemical structure (amino acid sequence) and also the correct secondary and tertiary structures (3 dimensional fold) be verified to assure safety and efficacy. Although numerous techniques are available to assess a biologic's primary, secondary and tertiary structures, difficulties arise when assessing higher order structure in the presence of protein excipients. In these studies far UV circular dichroism spectropolarimetry (far UV-CD) was used to determine the secondary structure of IFN alpha-2b in the presence of a protein excipient (bovine serum albumin, BSA). We demonstrated that the secondary structure of IFN alpha-2b remains mostly unchanged at a variety of BSA to IFN alpha-2b protein ratios. A significant difference in alpha helix and beta sheet content was noted when the BSA to IFN alpha-2b ratio was 5:1 (w/w), suggesting a potential conformational change in IFN alpha-2b secondary structure when BSA is in molar excess.

Approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography, gel electrophoresis and mass spectrometry.

A combination of separation and identification techniques was used to rapidly and reproducibly analyze influenza vaccine constituents. Size-exclusion HPLC analysis reduced significantly the complexity by providing a constituents profile according to size. Significantly, no sample treatment was required prior to analysis thus eliminating a potential source of artifacts and degradation. Distinct profiles were associated with influenza strains as well as with vaccines from different manufacturers. Samples analyzed over several years allowed evaluation of method performance and provided stability-indicating data relating to the structural integrity of separated components. Collected chromatographic peaks were identified by gel electrophoresis and MALDI/MS of tryptic digests from excised gel bands. The challenge in obtaining high quality analytical data from complex mixtures clearly demonstrated the value of separation steps prior to MS identification. The method presented here is not intended to replace existing methodology; it is intended to provide a product specific profile to be used as a rapid screen for manufacturer, year (for annual influenza vaccines), stability or counterfeit product. It is a new screening method that provides a rapid and robust indication of products which require further investigation as a result of a deviation in their characteristic profile. Until now this tool did not exist.