ABSTRACT
This report describes a new MHC class II allele, HLA-DR beta 1*0306, discovered in a 31-year-old Norwegian male. The allele typed serologically as DRw52 (DR3) and amplified in PCR using DR52-associated group primers. This product could not be identified using established restriction digests, however. Use of Asp 700, Msp I, Hha I, Bse RI, Mnl I, Hph I, and Bsrb I gave banding patterns expected for a DR beta 1*03011 allele, but Rsa I had an additional site at codon 47. Sequencing showed a single base change at this position, with the substitution of tyrosine for phenylanine in this new allele. The biologic impact of this substitution remains to be determined.
Subject(s)
Alleles , Genes, MHC Class II , HLA-DR Antigens/genetics , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Adult , Base Sequence , Codon/genetics , HLA-DRB1 Chains , Humans , Male , Molecular Sequence Data , Point Mutation , Sequence Alignment , Sequence Homology, Nucleic AcidABSTRACT
A simple economical procedure for purifying saxitoxin-induced protein (SIP) from crude extracts of the small shore crab, Hemigrapsus oregenesis, was developed. (NH4)2SO4 precipitation, chymotrypsin digestion, heat treatment, gel filtration and ion-exchange-chromatography procedures were evaluated in purifying SIP. An enzyme immunoassay was used to determine the SIP yield and relative purity at each step of three procedures, thus permitting an assessment of the conditions required for maximum recovery. Response surface analysis was used in an attempt to determine the optimum temperature and exposure time for the heat treatment. A 20 min incubation at 65 degrees C was confirmed by electrophoretic analysis to be the best combination of time and temperature for achieving both an acceptable yield and purity of SIP. SIP in desalted concentrate was shown to be resistant to chymotrypsin proteolysis; however, this enzyme had deleterious effects on SIP purification at later stages of the procedure. The omission of the chymotrypsin digestion, and the inclusion of gel-filtration chromatography in the final clean-up step, resulted in the purification of SIP comparable with that achieved with affinity chromatography.
