Strategy and implementation of a system for protein engineering.

This paper describes an overall view of an industrial protein engineering project from conception to successful completion. The choice of rational design was determined by the availability of an excellent three-dimensional crystal structure and the availability of information in the literature to define a strategy. The design strategy was refined extensively during the course of the project. The development of methods for mutagenesis, expression, verification, purification, and characterization of mutant enzymes is dictated in part by the enzyme property one chooses to modify and must be rapid yet accurate. Such an approach would be applicable to improve the stability of any other protein or enzyme. Using this approach, we successfully increased the stability of subtilisin BL over 10-fold at 50 degrees C with an overall success rate greater than 60%.

Developmental Regulation of beta-Conglycinin in Soybean Axes and Cotyledons.

Analysis of the expression of genes encoding the beta-conglycinin seed storage proteins in soybean has been used to extend our understanding of developmental gene expression in plants. The alpha, alpha', and beta subunits of beta-conglycinin are encoded by a multigene family which is organ-specific in its expression. In this study we report the differentially programmed accumulation of the alpha, alpha', and beta subunits of beta-conglycinin. Multiple isomeric forms of each subunit are present in the dry seed, but the timing of their accumulation is unique for each subunit. The previously reported variation in amount of alpha' and alpha subunits in axis and cotyledons is also reflected in the amount of subunit specific mRNA which is present in each tissue. The beta subunit, previously undetected in soybean axes, is found to be synthesized but rapidly degraded. These differences in beta-conglycinin protein accumulation may be reflected by the morphological differences observed in protein bodies between these two tissues.

Characterization of a cDNA encoding ricin E, a hybrid ricin-Ricinus communis agglutinin gene from the castor plant Ricinus communis.

Two classes of ricin cDNA clones have been identified and sequenced. The cDNA clone pBL-1 closely matches in nucleotide sequence the ricin genomic clone pAKG previously described by Halling et al., 1985 (Nucl. Acids Res. 13:8019). A second group of cDNA clones, represented by pBL-3, encode a hybrid protein (ricin E), having a ricin-like A chain and N-terminal half of the B chain and an RCA (Ricinus communis agglutinin)-like C-terminal half of the B chain.

Physical mapping of temporally regulated, overlapping transcripts in the region of the 10K protein gene in Autographa californica nuclear polyhedrosis virus.

We investigated transcriptional activity in the region of a gene for a major late protein (10 kilodaltons) of Autographa californica nuclear polyhedrosis virus. This 10K protein gene spans an HindIII cleavage site, with the 5' end of the gene located in the HindIII-Q fragment and the 3' end in the HindIII-P fragment. Northern blot analysis showed that there were at least four transcripts mapping in this region, two that are present maximally at 12 h (1,500 and 1,100 bases in length) and two that are present at 24 h (750 and 2,500 bases in length). Northern blot analysis also suggested that these transcripts overlap. S1 mapping identified the precise positions of the transcripts and confirmed the overlap. The 1,500- and 1,100-base transcripts share a common 5' end, about 1,056 bases upstream from the HindIII-P-Q boundary; the 750- and 2,500-base transcripts share a common 5' end, about 303 bases upstream from the HindIII-P-Q boundary. The 1,100-base transcript terminates in the Q fragment, about 234 bases from the HindIII-P-Q boundary. The other three transcripts all end in the P fragment. Both the 1,500- and the 750-base transcripts terminate approximately 240 bases from the HindIII-P-Q boundary. The 2,500-base transcript exhibits 3'-end heterogeneity, ending around 2,046 bases from the HindIII-P-Q boundary. Therefore, three different transcripts (1,500, 750, and 2,500 bases in length) contain the complete 10K protein coding region. A fourth transcript (1,100 bases in length) overlaps the 750-base 10K protein gene transcript by 70 bases.

Genomic cloning and characterization of a ricin gene from Ricinus communis.

A genomic clone that specifies a single polypeptide precursor for ricin, a toxic lectin of Ricinus communis (castor bean), was isolated, sequenced and Sl mapped. The gene encodes a 64 kDa precursor which contains, in the following order: a 24 or 35 amino acid signal peptide, the A chain, a 12 amino acid linker peptide, and the B chain. The 5'-end of the ricin mRNA maps approximately 35 bases upstream from the first methionine codon. Two putative TATA boxes and a possible CAAT box lie in the 5'-flanking region. Two possible polyadenylation signals were found in the 3' flanking region. No introns were found, which is typical of other lectin genes that have been sequenced. Southern blot analysis suggests that the castor bean genome contains approximately six ricin-like genes.

Molecular characterization of a deletion mutation affecting the alpha'-subunit of beta-conglycinin of soybean.

A naturally occurring variety of soybean Glycine max cv. Keburi, which lacks the alpha'-subunits of the 7S seed storage protein (beta-conglycinin), was recently described by Kitamura and Kaizuma. Keburi beta-conglycinins contain a normal complement of alpha-, beta-, and gamma-subunits that accumulate in a temporally and spatially regulated manner comparable to that of the standard cultivar Provar. Poly(A)+ RNA isolated from mid-to-late maturation stage Keburi seeds was translated in vitro, yielding products equivalent to those of Provar poly(A)+ RNA except that Keburi mRNA did not produce the pre-alpha'-subunit polypeptide. The basis of the Keburi phenotype was determined by examining genomic DNA using Southern blot hybridization. Restriction fragments isolated from a cloned 11.5 kb EcoRI fragment of genomic DNA containing a normal alpha'-subunit gene (Gmg 17.1) were used as hybridization probes. Sequences far upstream of the alpha'-subunit gene were present in both Provar and Keburi cultivars. However, hybridization reactions with probes from within the gene demonstrated that a deletion had occurred in Keburi DNA beginning immediately 5' to the alpha'-subunit gene represented on this 11.5 kb fragment. The deletion extends through most of the coding sequences, producing the Keburi phenotype.

Structural sequences are conserved in the genes coding for the alpha, alpha' and beta-subunits of the soybean 7S seed storage protein.

Cloned DNAs encoding four different proteins have been isolated from recombinant cDNA libraries constructed with Glycine max seed mRNAs. Two cloned DNAs code for the alpha and alpha'-subunits of the 7S seed storage protein (conglycinin). The other cloned cDNAs code for proteins which are synthesized in vitro as 68,000 d., 60,000 d. or 53,000 d. polypeptides. Hybrid selection experiments indicate that, under low stringency hybridization conditions, all four cDNAs hybridize with mRNAs for the alpha and alpha'-subunits and the 68,000 d., 60,000 d. and 53,000 d. in vitro translation products. Within three of the mRNA, there is a conserved sequence of 155 nucleotides which is responsible for this hybridization. The conserved nucleotides in the alpha and alpha'-subunit cDNAs and the 68,000 d. polypeptide cDNAs span both coding and noncoding sequences. The differences in the coding nucleotides outside the conserved region are extensive. This suggests that selective pressure to maintain the 155 conserved nucleotides has been influenced by the structure of the seed mRNA. RNA blot hybridizations demonstrate that mRNA encoding the other major subunit (beta) of the 7S seed storage protein also shares sequence homology with the conserved 155 nucleotide sequence of the alpha and alpha'-subunit mRNAs, but not with other coding sequences.

Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation.

The maturation of pseudorabies virus DNA from the replicative concatemeric form to molecules of genome length was examined using nine DNA+ temperature-sensitive mutants of pseudorabies virus, each belonging to a different complementation group. At the nonpermissive temperature, cells infected with each of the mutants synthesized concatemeric DNA. Cleavage of the concatemeric DNA to genome-length viral DNA was defective in all the DNA+ ts mutants tested, indicating that several viral gene products are involved in the DNA maturation process. In none of the ts mutant-infected cells were capsids with electron-dense cores (containing DNA) formed. Empty capsids with electron-translucent cores were, however, formed in cells infected with six of the nine temperature-sensitive mutants; in cells infected with three of the mutants, no capsid assembly occurred. Because these three mutants are deficient both in maturation of DNA and in the assembly of viral capsids, we conclude that maturation of viral DNA is dependent upon the assembly of capsids. In cells infected with two of the mutants (tsN and tsIE13), normal maturation of viral DNA occurred after shiftdown of the cells to the permissive temperature in the presence of cycloheximide, indicating that the temperature-sensitive proteins involved in DNA maturation became functional after shiftdown. Furthermore, because cycloheximide reduces maturation of DNA in wild-type-infected cells but not in cells infected with these two mutants, we conclude that a protein(s) necessary for the maturation of concatemeric DNA, which is present in limiting amounts during the normal course of infection, accumulated in the mutant-infected cells at the nonpermissive temperature. Concomitant with cleavage of concatemeric DNA, full capsids with electron-dense cores appeared after shiftdown of tsN-infected cells to the permissive temperature, indicating that there may be a correlation between maturation of DNA and formation of full capsids. The number of empty and full capsids (containing electron-dense cores) present in tsN-infected cells incubated at the nonpermissive temperature, as well as after shiftdown to the permissive temperature in the presence of cycloheximide, was determined by electron microscopy and by sedimentation analysis in sucrose gradients. After shiftdown to the permissive temperature in the presence of cycloheximide, the number of empty capsids present in tsN-infected cells decreased with a concomitant accumulation of full capsids, indicating that empty capsids are precursors to full capsids.