Pichia surface display: display of proteins on the surface of glycoengineered Pichia pastoris strains

Expression of proteins on the surface of yeasts has a wide range of applications in biotechnology, such as directed evolution of proteins for increased affinity and thermal stability, screening of antibody libraries, epitope mapping, and use as whole-cell biocatalysts. However, hyperglycosylation can interfere with overall protein accessibility on the surface. Therefore, the less elaborate hyperglycosylation in wild type Pichia pastoris and the availability of glycoengineered strains make this yeast an excellent alternative for surface display of glycoproteins. Here, we report the implementation of the well-established a-agglutinin-based yeast surface display technology in P. pastoris. Four heterologous proteins were expressed on the surface of a wild type and a glycoengineered strain. Surface display levels were monitored by Western blot, immunofluorescence microscopy, and FACS analysis. The availability of glycoengineered strains makes P. pastoris an excellent alternative for surface display of glycoproteins and paves the way for new applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel expression vector, designated as pHisJM, for producing recombinant His-fusion proteins

Compared to glutathione S -transferase (GST), tagging with hexahistidine residues (His) has several merits: low levels of toxicity and immunogenicity, a smaller size and no electric charge. We have constructed a novel expression vector, designated as pHisJM (EMBL/GenBank/DDJB accession no. AB116367), for producing recombinant His-fusion proteins. This vector was constructed by replacing GST and multiple cloning site (MCS) cassettes in pGEX-5X-3 with those of hexahistidine and MCS derived from pRSET C vector. Human annexin IV (Anx IV) was used as target protein. His-Anx IV fusion protein was expressed using pHisJM and gave a 40 kDa band when immuno-stained with anti-His mAb or anti-Anx IV mAb as predicted. To compare expression efficiency, a Anx IV cDNA inserted-pHisJM or pGEX-5X-3 was transformed into Escherichia coli DH5alpha, JM109, BL21 and BL21(DE3). Using pHisJM, Anx IV protein was highly expressed in all cell strains. In addition to the merits of using His-tag, pHisJM has several advantages: 1) it has high expression efficiency; 2) it can be used in any Escherichia coli strain; and 3) it can be used in a single strain of Escherichia coli in all steps from plasmid construction to the expression of the target gene.     

Construction of Escherichia coli dnaK-deletion mutant infected by lambdaDE3 for overexpression and purification of recombinant GrpE proteins

Escherichia coli is widely employed to produce recombinant proteins because this microorganism is simple to manipulate, inexpensive to culture, and of short duration to produce a recombinant protein. However, contamination of molecular chaperone DnaK during purification of the recombinant protein is sometimes a problem, since DnaK sometimes has a negative effect on subsequent experiments. Previously, several efforts have been done to remove the DnaK contaminants by several sequential chromatography or washing with some expensive chemicals such as ATP. Here, we developed a simple and inexpensive method to express and purify recombinant proteins based on an E. coli dnaK-deletion mutant. The E. coli ΔdnaK52 mutant was infected by λDE3 phage to overexpress desired recombinant proteins under the control of T7 promoter. Using this host cell, recombinant hexa histidine-tag fused GrpE, which is well known as a co-chaperone for DnaK and to strongly interact with DnaK, was overexpressed and purified by one-step nickel affinity chromatography. As a result, highly purified recombinant GrpE was obtained without washing with ATP. The purified recombinant GrpE showed a folded secondary structure and a dimeric structure as previous findings. In vitro ATPase activity assay and luciferase-refolding activity assay demonstrated that the recombinant GrpE worked together with DnaK. Thus, this developed method would be rapid and useful for expression and purification of recombinant proteins which is difficult to remove DnaK contaminants.     

Selection of Burkholderia pseudomallei protease-binding peptides by phage display

Burkholderia pseudomallei is a causative agent of melioidosis, a fatal community acquired septicemia in Southeast Asia and Northern Australia. A protease has been proposed to be one of the major pathogenic factors to play a significant role in melioidosis. We have used phage display technology to identify peptides binding to B. pseudomallei protease. By screening a constrained cyclic heptapeptide library, five independent clones with affinity to this protease were isolated and the amino acid sequences were determined. The cyclic heptapeptides from two of the phage clones (Cys-Phe-Phe-Met-Pro-His-Thr-Phe-Cys) were identical and showed the strongest phage-protease interaction as detected by ELISA. Four of the five selected phages at the amount of 10¹³ phages could inhibit B. pseudomallei protease activity by approximately 50%.     

Functional expression and display of an antibody Fab fragment in Escherichia coli: study of vector designs and culture conditions

Several different vector designs are currently being used to display and express Fab molecules in Escherichia coli, but their relative efficiency in phage display and protein expression cannot be compared from the published data. We systematically investigated which vector design most effectively displays and expresses Fab molecules in E. coli using, as a model system, a human Fab against tetanus toxoid (tt). Three different vectors were used in this study: pFab1 where the VL-CL and VH-CH1 genes were driven by two promoters in two separate expression cassettes, and pFab2 and pFab3 that both contain one dicistronic expression cassette with two translation initiation sites and either VH-CH1 before VL-CL or VL-CL before VH-CH1, respectively. The display of tt-Fab on the surface of phage and the expression of tt-Fab protein in E. coli were compared for the aforementioned vectors. Our results showed that the pFab3 vector was most effective in Fab display. A 10-fold increase in the expression of secreted Fab was observed in pFab3 when compared with vectors pFab1 and pFab2. Further experiments were conducted using pFab3 to optimize expression levels using different strains of E. coli and various culture conditions. The highest expression of tt-Fab was obtained using the pFab3 vector in host strain JM105 with an induction temperature at 37 degrees C and IPTG concentration of 0.1 mM.     

A phage display system for studying the sequence determinants of protein folding.

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.     

Physiological characteristics of recombinant Escherichia coli cells displaying poly-His peptides

Using the Escherichia coli OmpC protein as an anchoring motif, four different poly-His units (1, 2, 3 and 6 copies of 6-His) were displayed on the seventh loop of the OmpC. Recombinant E. coli strains displaying 1, 3 or 6 copies of poly-His became much more sensitive to SDS (0.1%, w/v) and EDTA (2 mM) compared with control strains. However, recombinant E. coli cells displaying 2 copies of poly-His were resistant to SDS and EDTA; greater than 70% and 90% of cells maintained cell integrity after 60 min treatment with SDS and EDTA, respectively, suggesting its usefulness as a whole cell biosorbent.     

Protein folding, misfolding, and refolding of therapeutic proteins

Substantial progress has been made towards understanding the folding mechanisms of proteins in vitro and in vivo even though the general rules governing such folding events remain unknown. This paper reviews current folding models along with experimental approaches used to elucidate the folding pathways. Protein misfolding is discussed in relation to disease states, such as amyloidosis, and the recent findings on the mechanism of converting normally soluble proteins into amyloid fibrils through the formation of intermediates provide an insight into understanding the pathogenesis of amyloid formation and possible clues for the development of therapeutic treatments. Finally, some commonly adopted refolding strategies developed over the past decade are summarized.     

Optimizing synthesis and expression of transmembrane peptides and proteins

Structural studies of full-length membrane proteins have been hindered by their hydrophobicity and low expression in a variety of systems. However, a simplifying aspect of membrane protein folding is that individual transmembrane segments or membrane protein fragments have been observed to represent independent folding domains, and as such, can facilitate the study of packing interactions between TM helices, and the collection of structural information regarding membrane proteins. This review focuses on two categories of techniques--total peptide synthesis and bacterial expression--that can each be optimized for preparation of transmembrane protein segments. First, synthesis of hydrophobic transmembrane peptides that are N- and/or C-tagged with solubilizing residues such as lysine can improve manipulation of the transmembrane core in a variety of biophysical experiments. In this context, we describe general protocol considerations during the synthesis, cleavage, and purification stages of these peptides to identify appropriate parameters that combine to improve yields of hydrophobic peptides. Second, bacterial expression of membrane protein fragments is a useful tool for producing large quantities of hydrophobic protein segments. Targeting protein expression within Escherichia coli can facilitate purification, while attaching the hydrophobic construct to a hydrophilic fusion protein can amplify expression. We show that adapting protein constructs to comply with expression host specifications, in concert with thorough exploration of expression conditions such as the type of media used for expression, temperature, and cell strain, can significantly improve protein yields.     

Use of recA803, a partial suppressor of recF, to analyze the effects of the mutant Ssb (single-stranded DNA-binding) proteins in vivo and in vitro

Summary We examined the possibility that the ssb-1 and ssb-113 mutants exert some of their effects by interfering with the normal function of wild-type RecF protein. Consistent with this possibility, we found that recA803, which partially suppresses recF mutations, also partially suppresses both ssb mutations, as detected by an increase in UV resistance. No evidence was obtained for suppression of the defect in lexA regulon inducibility caused by the ssb mutations. Consequently we suggest that suppression occurs by increasing recombinational repair. In vitro tests of Ssb mutant and wild-type proteins revealed that the single-stranded DNA dependent ATPase activity of RecA protein is more susceptible to inhibition than the joint-molecule-forming activity. All three Ssb proteins inhibit the ATPase activity of RecA wild-type protein almost completely while under similar conditions they inhibit the joint-molecule-forming activity only slightly. Both activities of RecA803 protein were found to be less inhibited by the three Ssb proteins than those of RecA wild-type protein. This is consistent with the suppressing ability of recA803. We found no evidence to contradict the previously proposed hypothesis that ssb-1 affects recombinational repair by acting as a weaker form of Ssb protein. We found, however, only very weak evidence that Ssb-113 protein interferes directly with recombinational repair so that the possibility that it interferes with a normal function of RecF protein must remain open.