J. Bruce Caldwell

Low molecular weight albumins from sunflower seed: identification of a methionine-rich albumin

Abstract The small M r , proteins of sunflower seed ( Helianthus annuus ) are soluble in 60% (by vol) methanol. These proteins, classified as albumins on the basis of their solubility in water, were isolated from a salt extract of sunflower seed by precipitating the 11S globulins with 60% (by vol) methanol and were resolved into eight distinct components by reversed-phase HPLC. Electrophoresis showed that each fraction contained a single polypeptide chain with an apparent M r , in the range 10 000–18 000. The individual sunflower albumins are basic proteins with distinct amino acid compositions. The major albumins (4–8) contain high contents of glutamine/glutamic acid, asparagine/aspartic acid, arginine and cysteine, characteristic of the 2S class of seed storage proteins. One exception was the small glutamine/glutamic acid content of albumin 6. Two of the sunflower albumins (7 and 8) with M r - 10 000 were methionine-rich proteins containing 16 residues per cent methionine as well as eight residues per cent cysteine. These sulphur-rich proteins constitute some 7% of the total salt extractable seed protein. A method for the preparation of these two albumins using a reversed-phase Sep-pak cartridge is described.

Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41

The VH domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAGTM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The VH-FLAG was composed of three isoforms (pI values of ∼4.6, 4.9, and 5.3) and the VH molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the VH isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the VH domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its1H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the VH molecule, prevented dimerization of the VH and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.

Sunflower 11S globulin, susceptibility to proteolytic cleavage of the subunits of native helianthinin during isolation: HPLC fractionation of the subunits

Abstract The subunits of helianthinin, the major sunflower seed globulin, are susceptible to proteolytic cleavage or nicking of the acidic polypeptide chains by endogenous seed proteases during isolation. The course of the degradation was determined using reducing and non-reducing SDS-PAGE. This degradation can be partly retarded by including high salt and the protease inhibitor, phenylmethylsulphonyl fluoride in the extraction buffer. Separation of the globulins and albumins stopped the degradation of the helianthinin subunits. Rapid precipitation of the sunflower globulins with 60% methanol was the most effective way of isolating helianthinin with undegraded subunits. Native helianthinin was also extensively degraded by trypsin and chymotrypsin. Helianthinin was dissociated into its component subunits in 0.1 % trifluoroacetic acid and these were separated by reversed-phase HPLC. Eleven discrete fractions were obtained and analysed by SDS-PAGE. Six distinct subunits of M r

Amino acid sequence of the acidic kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC]

56 000 and

Primary structure of kunitz-type trypsin inhibitor-2a (pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed

52 000 and one subunit of

Recombinant anti-sialidase single-chain variable fragment antibody. Characterization, formation of dimer and higher-molecular-mass multimers and the solution of the crystal structure of the single-chain variable fragment/sialidase complex.

46 000 were identified. Three fractions contained essentially a single subunit, composed of two disulphide-linked polypeptide chains, suitable for further structural analysis. The amino acid compositions of the separated subunits of helianthinin are presented.

Amino acid and cDNA sequences of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L.)

The primary sequence of trypsin inhibitor-2 (WBTI-2) fromPsophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 fromErythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors fromAdenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that thePsophocarpus andErythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.

Recombinant antineuraminidase single chain antibody: expression, characterization, and crystallization in complex with antigen.

The primary structure of acidic trypsin inhibitor-2a (WBTI-2a,pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed was determined. This inhibitor consists of a single polypeptide chain of 180 amino acids including four half-cystine residues and has an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2a,pI 5.9, showed 84% identity to acidic trypsin inhibitor-2 (WBTI-2,pI 5.1) but only 57% identity to the basic trypsin inhibitor (WBTI-1,pI 8.9) and 50% identity to the chymotrypsin inhibitor of winged bean. The data indicate that winged bean seed contains a family of three Kunitz-type inhibitors which have about 50% identity.