BackgroundBioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale.ResultsHere, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase® technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor.ConclusionsCultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first screening phase not only shortens the developmental line by guarantying that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.
Type II restriction endonucleases (REases) form a large and highly diverse group of enzymes. Even REases specific for a common recognition site often vary in their oligomeric structure, domain organization and DNA cleavage mechanisms. Here we report biochemical and structural characterization of the monomeric restriction endonuclease UbaLAI, specific for the pseudosymmetric DNA sequence 5′-CC/WGG-3′ (where W = A/T, and ‘/’ marks the cleavage position). We present a 1.6 Å co-crystal structure of UbaLAI N-terminal domain (UbaLAI-N) and show that it resembles the B3-family domain of EcoRII specific for the 5′-CCWGG-3′ sequence. We also find that UbaLAI C-terminal domain (UbaLAI-C) is closely related to the monomeric REase MvaI, another enzyme specific for the 5′-CCWGG-3′ sequence. Kinetic studies of UbaLAI revealed that it requires two recognition sites for optimal activity, and, like other type IIE enzymes, uses one copy of a recognition site to stimulate cleavage of a second copy. We propose that during the reaction UbaLAI-N acts as a handle that tethers the monomeric UbaLAI-C domain to the DNA, thereby helping UbaLAI-C to perform two sequential DNA nicking reactions on the second recognition site during a single DNA-binding event. A similar reaction mechanism may be characteristic to other monomeric two-domain REases.
Nearly 3000 restriction endonucleases with over 200 different specificities, which together with cognate DNA methyltransferases constitute restriction-modification (R-M) 1 systems, have been identified in bacteria (1). Restrictionmodification enzymes are traditionally divided into three classes designated type I, II, and III on the basis of enzyme subunit composition, cofactor requirements, substrate specificity characteristics, and reaction products (2). An increasing number of restriction endonucleases that do not fit into the conventional classification however have been reported (3-7). Their differences from the type I and type III enzymes are so substantial that a classification as new kinds of restriction endonucleases; type IIS, type IIT, type IV, and Bcg-like has been suggested (3-8).The type IV restriction endonuclease Eco57I has been studied in detail (4). Similar to type IIS endonucleases, it recognizes an asymmetric nucleotide sequence, cleaves both DNA strands outside the target site 5Ј-CTGAAG(N) 16/142 and exists in solution as a monomer. Other features of Eco57I, however, such as stimulation of endonucleolytic reaction with the DNA methyltransferase cofactor S-adenosyl-L-methionine (AdoMet) and methylation of one strand of the recognition duplex, makes it similar to type III enzymes. Both endonucleolytic and methylation activities reside within a single large polypeptide of the enzyme. In addition to the bifunctional restriction endonuclease, the Eco57I R-M system also includes a separate Eco57I methyltransferase, which modifies both DNA strands of the target duplex. The methylation domain has been previously assigned to the carboxyl-half of the Eco57I restriction endonuclease, where conserved amino acid sequence motifs typical for m 6 A DNA methyltransferases involved in AdoMet binding and catalysis of methyl group transfer are located (9). The location and identity of the endonuclease active center though remains to be determined and is addressed here.In contrast to DNA methyltransferases, the amino acid sequences of restriction endonucleases share little similarity. This observation therefore reduces the possibility of identifying catalytic sites of restriction enzymes on the basis of sequence alignment. Structural and mutational analysis of type II restriction endonucleases revealed however the PDX n (D/E)XK motif as a catalytic/Mg 2ϩ binding signature motif (8, 10, 11). Two putative catalytic/Mg 2ϩ binding motifs (i.e. 77 PDX 13 EAK and 811 PDX 20 DQK, located in the N-terminal and C-terminal parts of Eco57I, respectively) have been described in the amino acid sequence of the enzyme (10). The statistical significance of these motifs however is low, and their presence does not allow unambiguous prediction of the active site, as is evidenced by the following observations. (i) Cfr10I contains the PDX n (D/ E)XK motif, but it is not part of its catalytic center (12) and (ii) EcoRI contains two such motifs, one of which is not involved in catalysis (10).On the other hand it cannot be excluded that...
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